Compositions and methods for activating innate and allergic immunity

ABSTRACT

Methods for making and using therapeutic formulations of Proteosome-based immunoactive compositions are provided. The immunogenic compositions, which include Proteosomes and liposaccharides, may be used to elicit or enhance a nonspecific innate immune response to, for example, treat or prevent infectious disease. In addition, after activating the innate immune system, immunogenic compositions further containing an antigen may be used to elicit a specific adaptive immune response. Furthermore, provided are compositions capable of altering hyperreactive responses or inflammatory immune responses, such as allergic reactions. Such compositions may be used as a prophylactic, or in various clinical settings to treat or prevent infectious disease (such as parasite, fungal, bacterial or viral infections), or to alter inappropriate inflammatory immune responses (such as allergic reactions or asthma).

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.10/972,062, filed Oct. 22, 2004, now pending; which claims the benefitof U.S. Provisional Patent Applications No. 60/513,614 filed Oct. 22,2003 and No. 60/559,842 filed Apr. 6, 2004, all of which applicationsare incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to immunomodulation and, morespecifically, to therapeutic uses of immunostimulatory Proteosomecompositions for inducing a nonspecific immune response (such as aninnate immune response) so that an adaptive immune response ispotentiated or enhanced, or to induce both a nonspecific immune responseand a specific adaptive immune response, such that infectious disease istreated or prevented, or to modulate an immune response for treating orpreventing an inflammatory reaction, such as allergic asthma.

2. Description of the Related Art

Some microbial pathogens are capable of causing fatal infections evenwhen faced with a robust host immune response. Nonetheless, control oframpant infectious disease has been generally successful in modemsociety by using strict public health measures, drugs (such asantibiotics), and vaccines. Vaccines typically include an attenuatedmicrobe or a microbial antigen to activate a specific (adaptive) immuneresponse. The ability of an antigen to induce a protective immuneresponse in a host can be enhanced by formulating the antigen with animmunostimulant or an adjuvant. Alum-based adjuvants are almostexclusively used for licensed, injectable human vaccines. However, theadaptive immune response requires signals that provide information aboutthe origin of the antigen (i.e., self versus non-self antigens) and thetype of response to be induced (i.e., a T cell and/or B cell response).Evidence recently accumulated indicates that these signals may beprovided by the innate immune system (see, e.g., Fearon and Locksley,Science 272:50, 1996; Medzhitov and Janeway, Curr. Opin. Immunol. 9:4,1997).

Innate immunity is the first line of antibody-independent defenseagainst infections and, in many instances, can eliminate infectiousagents. The components of innate immunity recognize structures that arecharacteristic of microbial pathogens and are not present on mammaliancells. The principle effector cells of innate immunity are neutrophils,mononuclear phagocytes, and natural killer (NK) cells. Neutrophils andmacrophages express surface receptors that recognize microbes in theblood and tissues, and either stimulate the ingestion (phagocytosis,e.g., mannose or opsonin receptors) or activate phagocytes not involvedin ingestion (e.g., Toll-like receptors, TLRs). The effector mechanismsof innate immunity are often used to eliminate microbes, even in anadaptive immune response. Thus, the innate immune response can providesignals that function in concert with antigen to stimulate theproliferation and differentiation of antigen-specific (adaptive) T and Blymphocytes.

An efficient immune response depends on the communication between theinnate and adaptive immune responses. The T lymphocyte is important forcoordinating the adaptive immune response by controlling the release ofeffector molecules. For example, T helper (Th) 1 cells produceinterleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), andinterferon gamma (IFN-γ), which are important for the development ofcell-mediated immunity (Mosmann et al., J. Immunol. 136: 2348, 1986;Street and Mosmann, FASEB J. 5: 171, 1991). In contrast, Th2 cellsproduce IL-4, IL-13, IL-5, IL-9, IL-6 and IL-10, which are important forthe stimulation of IgE production, mucosal mastocytosis, andeosinophilia (Mosmann et al.; Street and Mosmann). While a shift towarda Th1 or Th2 phenotype may be important for the defense againstpathogens, a shift in one direction or another can also be associatedwith the induction of autoimmune disease (Th1) or inflammatory disease(Th2).

In inflammatory diseases, such as allergy or asthma, the fine balancebetween the Th1, Th2 and T regulatory cytokine responses appears toshift toward a Th2 phenotype. For example, asthma is a complexinflammatory disease of the lung characterized by variable airflowobstruction, airway hyperresponsiveness (AHR), and airway inflammation.Although asthma is multifactorial in origin, the inflammatory process(in the most common form of asthma, referred to as extrinsic or allergicasthma) is believed to be the result of an abnormal immune response tocommonly inhaled allergens. The presentation of inhaled allergens toCD4+ T cells in the lungs of susceptible individuals results in theproduction of Th2 cytokines, IL-4, IL-13 and IL-5, which control thedifferentiation, recruitment, and activation of mast cells andeosinophils in the airway mucosa. These effector cells release a varietyof inflammatory mediators (e.g., histamine, mucous secretogues,eosinophil-derived basic proteins, proteases). The mediators eitherindividually or in concert cause acute bronchoconstriction, disruptionof the airway epithelial layer, alterations in neural control of airwaytone, increased mucus production, and increased smooth muscle cell mass.Each of these consequences of the inflammatory process may cause oroccur in combination with AHR. The incidence, morbidity, and mortalityof asthma has increased worldwide over the last two decades, and theexisting anti-inflammatory medications (such as corticosteroids) havelimitations in that the disease is not modified (i.e., only the symptomsare treated, which will return if the medication is discontinued) andthese medications are associated with the potential for significant sideeffects.

Hence, a need exists for identifying and developing immunostimulatorycompositions that are therapeutically effective against microbialinfections and immunopathologic (e.g., inflammatory) responses to suchinfections, particularly compositions that can potentiate or enhanceprotective immunity, and compositions that can suppress animmunopathologic response. The present invention meets such needs, andfurther provides other related advantages.

BRIEF SUMMARY OF THE INVENTION

The invention described herein provides methods for making and usingtherapeutic formulations of Proteosome-based immunoactive compositions.Immunostimulatory compositions, which include Proteosomes andliposaccharides, may be used to elicit or enhance a nonspecific innateimmune response to treat or prevent infectious disease. In addition,after activating the innate immune system, immunogenic compositionsfurther containing an antigen may be used to elicit a specific adaptiveimmune response. Furthermore, provided are compositions capable ofaltering hyperreactive responses or inflammatory immune responses, suchas allergic reactions.

In one embodiment of the invention is provided a method for eliciting anonspecific immune response, comprising administering to a subject animmunostimulatory composition in an amount sufficient to elicit anonspecific immune response, wherein the immunostimulatory compositioncomprises Proteosomes and liposaccharide. In one embodiment, thenonspecific immune response is an innate immune response that preventsor treats a microbial infection, wherein the microbial infection is aviral, parasitic, fungal, or bacterial infection. In a particularembodiment, the microbial infection is a viral infection, wherein theviral infection is an influenza infection. In one embodiment, theimmunostimulatory composition is administered by a route selected fromat least one of mucosal, enteral, parenteral, transdermal, transmucosal,nasal, and inhalation. In an embodiment, the liposaccharide finalcontent by weight as a percentage of Proteosome protein ranges fromabout 1% to 500%. In certain embodiments, the proteosomes andliposaccharide are obtained from the same Gram-negative bacterialspecies, or the proteosomes are obtained from a first Gram-negativebacterial species and the liposaccharide is obtained from a secondGram-negative bacterial species. In a particular embodiment, theliposaccharide is obtained from a Gram-negative bacterium selected fromat least one of Shigella species, Chlamydia species, Yersinia species,Pseudomonas species, Plesiomonas species, Escherichia species,Porphyromonas species, and Salmonella species. In a particularembodiment, the Proteosomes are obtained from Neisseria species, and inanother particular embodiment, the Proteosomes are obtained fromNeisseria meningitidis, and the liposaccharide is obtained from Shigellaflexneri.

In another embodiment, the method provided further comprisesadministering to the subject an immunogenic composition afteradministering the immunostimulatory composition, wherein the immunogeniccomposition comprises Proteosomes, liposaccharide, and a microbialantigen, wherein the microbial antigen is a viral antigen, a bacterialantigen, a fungal antigen, or a parasitic antigen. In a particularembodiment, the microbial antigen is recombinant. In other embodiments,the microbial antigen is a bacterial antigen obtained from Bacillusanthracis, Chlamydia trachomatis, Yersinia pestis, or EnteropathogenicEscherichia coli. In a particular embodiment, the bacterial antigen isProtective Antigen from Bacillus anthracis. In another particularembodiment, the microbial antigen is a viral split antigen, wherein theviral split antigen is an influenza split antigen. In certainembodiments, the immunogenic composition comprises at least twomicrobial antigens, which may be obtained from the same microorganism,which is a virus, bacteria, fungus, or protozoa, or may be obtained fromdifferent microorganisms. The immunogenic composition elicits anadaptive immune response according to certain embodiments. According toparticular embodiments, the ratio of the weight of Proteosomes andliposaccharide of the immunogenic composition to the weight of themicrobial antigen of the immunogenic composition is within a range from4:1 to 1:4, 1:1 to 1:500, or 1:1 to 1:200. In other embodiments, theimmunogenic composition is administered about one to about ten days orabout one to seven days after the immunostimulatory composition, and inother certain embodiments, at least one of the immunostimulatorycomposition and the immunogenic composition further comprise apharmaceutically acceptable carrier.

The invention also provides a method for treating or preventing amicrobial infection, comprising (a) administering to a subject animmunostimulatory composition, wherein the immunostimulatory compositioncomprises Proteosomes and liposaccharide, in an amount and underconditions sufficient to elicit an innate immune response; and (b)administering to the subject an immunogenic composition, wherein theimmunogenic composition comprises Proteosomes, liposaccharide, and amicrobial antigen, in an amount and under conditions sufficient toelicit an adaptive immune response, such that the microbial infection istreated or prevented, wherein the microbial infection is a viral,parasitic, fungal, or bacterial infection. In a particular embodiment,the microbial infection is a viral infection, wherein the viralinfection is an influenza infection. In a particular embodiment, theimmunostimulatory composition is administered about one to about tendays before the immunogenic composition. In one embodiment, each of theimmunostimulatory composition and the immunogenic composition isadministered by a route selected from at least one of mucosal, enteral,parenteral, transdermal, transmucosal, nasal, and inhalation; and in aparticular embodiment the compositions are administered nasally.According to one embodiment, the liposaccharide final content by weightas a percentage of Proteosome protein ranges from about 1% to 500% ineach of the immunostimulatory and immunogenic compositions. Inparticular embodiments, the Proteosomes and the liposaccharide of theimmunostimulatory composition are obtained from the same Gram-negativebacterial species or the Proteosomes and the liposaccharide of theimmunostimulatory composition are obtained from the differentGram-negative bacterial species. In another particular embodiment, theProteosomes and the liposaccharide of the immunogenic composition areobtained from the same Gram-negative bacterial species, or theProteosomes and the liposaccharide of the immunogenic composition areobtained from different Gram-negative bacterial species. In otherparticular embodiments, the Proteosomes of the immunostimulatory andimmunogenic compositions are obtained from Neisseria species, and atleast one of the liposaccharides of the immunostimulatory andimmunogenic compositions is obtained from at least one of Shigellaspecies, Chlamydia species, Yersinia species, Pseudomonas species,Plesiomonas species, Escherichia species, Porphyromonas species, andSalmonella species. In another specific embodiment, the Proteosomes ofeach of the immunostimulatory and immunogenic compositions are obtainedfrom Neisseria meningitidis, and the liposaccharide of each of theimmunostimulatory and immunogenic compositions is from Shigellaflexneri. the microbial antigen is a viral antigen, a bacterial antigen,a fungal antigen, or a parasitic antigen. In a particular embodiment,the microbial antigen of the immunogenic composition is recombinant. Inother embodiments, the microbial antigen is a bacterial antigen obtainedfrom Bacillus anthracis, Chlamydia trachomatis, Yersinia pestis, orEnteropathogenic Escherichia coli. In a particular embodiment, thebacterial antigen is Protective Antigen from Bacillus anthracis. Inanother particular embodiment, the microbial antigen is a viral splitantigen, wherein the viral split antigen is an influenza split antigen.In certain embodiments, the immunogenic composition comprises at leasttwo microbial antigens, which may be obtained from the samemicroorganism, which is a virus, bacteria, fungus, or protozoa, or maybe obtained from different microorganisms. The immunogenic compositionelicits an adaptive immune response according to certain embodiments.The immunogenic composition elicits an adaptive immune responseaccording to certain embodiments. According to particular embodiments,the ratio of the weight of Proteosomes and liposaccharide of theimmunogenic composition to the weight of the microbial antigen of theimmunogenic composition is within a range from 4:1 to 1:4, 1:1 to 1:500,or 1:1 to 1:200. In other embodiments, the immunogenic composition isadministered about one to seven days or about one to about ten daysafter the immunostimulatory composition, and in other certainembodiments, at least one of the immunostimulatory composition and theimmunogenic composition further comprise a pharmaceutically acceptablecarrier.

Also provided is a method for altering an inflammatory immune response,comprising administering to a subject an immunomodulatory composition inan amount sufficient to alter an inflammatory immune response, whereinthe immunomodulatory composition comprises Proteosomes and aliposaccharide, wherein the immunomodulatory composition is administeredby a route selected from at least one of mucosal, enteral, parenteral,transdermal, transmucosal, nasal, and inhalation. In a particularembodiment, the liposaccharide final content by weight as a percentageof Proteosome protein ranges from about 1% to 500%. In a certainembodiment, the Proteosomes and liposaccharide are obtained from thesame Gram-negative bacterial species, and in another certain embodiment,the Proteosomes and liposaccharide are obtained from differentGram-negative bacterial species. The Gram-negative bacterial species,according to certain embodiments, is selected from at least one ofShigella species, Chlamydia species, Yersinia species, Pseudomonasspecies, Plesiomonas species, Escherichia species, Porphyromonas sp.,and Salmonella species. In a particular embodiment, the Proteosomes areobtained from Neisseria species. In another particular embodiment, theProteosomes are obtained from Neisseria meningitidis and theliposaccharide is obtained from Shigella flexneri.

In one embodiment, the method for altering an inflammatory immuneresponse, comprising administering to a subject an immunomodulatorycomposition in an amount sufficient to alter an inflammatory immuneresponse, wherein the immunomodulatory composition comprises Proteosomesand a liposaccharide further comprises administering to the subject animmunogenic composition after administering the immunomodulatorycomposition, wherein the immunogenic composition comprises Proteosomes,a liposaccharide, and an antigen. In a certain embodiment, theimmunogenic composition comprises at least one microbial antigen,wherein the at least one microbial antigen is viral, bacterial, fungal,or parasitic. According to particular embodiments, the ratio of theweight of Proteosomes and liposaccharide of the immunogenic compositionto the weight of the microbial antigen of the immunogenic composition iswithin a range from 4:1 to 1:4, 1:1 to 1:500, or 1:1 to 1:200. In aparticular embodiment, the antigen of the immunogenic composition isrecombinant. In another embodiment, the antigen of the immunogeniccomposition is bacterial, which bacterial antigen is obtained fromBacillus anthracis, Chlamydia trachomatis, Yersinia pestis, orEnteropathogenic Escherichia coli. In a certain embodiment, thebacterial antigen is Protective Antigen from Bacillus anthracis. Inanother certain embodiment, the antigen of the immunogenic compositionis a viral split antigen, and in a particular embodiment, the viralsplit antigen is an influenza split antigen. In another embodiment, theimmunogenic composition is administered about one to about ten daysafter the immunomodulatory composition, and in another particularembodiment, the immunogenic composition elicits an adaptive immuneresponse. In certain particular embodiments, the inflammatory immuneresponse is asthma or an allergic reaction. According to a particularembodiment, at least one of the immunomodulatory composition and theimmunogenic composition further comprises a pharmaceutically acceptablecarrier.

Also provided is a method for treating or preventing an allergicreaction, comprising (a) administering to a subject in need thereof animmunomodulatory composition, wherein the immunomodulatory compositioncomprises Proteosomes and a liposaccharide, in an amount and underconditions sufficient to alter an inflammatory immune response; and (b)administering to the subject an immunogenic composition, wherein theimmunogenic composition comprises Proteosomes, liposaccharide, and anallergen, in an amount and under conditions sufficient to elicittolerance to the allergen, such that the allergic reaction is treated orprevented, wherein each of the immunomodulatory composition and theimmunogenic composition is administered by a route selected from atleast one of mucosal, enteral, sublingual, parenteral, transdermal,transmucosal, nasal, and inhalation. In a particular embodiment, theimmunomodulatory composition is administered about one to about ten daysbefore the immunogenic composition. In one particular embodiment, theliposaccharide final content by weight as a percentage of Proteosomeprotein ranges from about 1% to 500% in each of the immunomodulatory andimmunogenic compositions. In one embodiment, the Proteosomes andliposaccharide of the immunomodulatory composition are obtained from thesame Gram-negative bacterial species, and in another embodiment, theProteosomes and liposaccharide of the immunomodulatory composition areobtained from different Gram-negative bacterial species. In anotherparticular embodiment, the Proteosomes and liposaccharide of theimmunogenic composition are obtained from the same Gram-negativebacterial species, and in still another particular embodiment, theProteosomes and liposaccharide of the immunogenic composition areobtained from different Gram-negative bacterial species. In oneparticular embodiment, the Proteosomes of each of the immunomodulatoryand immunogenic compositions are obtained from Neisseria species and theliposaccharide of at least one of the immunomodulatory composition andthe immunogenic composition is obtained from at least one of Shigellaspecies, Chlamydia species, Yersinia species, Pseudomonas species,Plesiomonas species, Escherichia species, Porphyromonas species, andSalmonella species. In a certain particular embodiment, the Proteosomesof each of the immunomodulatory and immunogenic compositions areobtained from Neisseria meningitidis, and the liposaccharide of each ofthe immunomodulatory and immunogenic compositions is obtained fromShigella flexneri. In another embodiment, the immunogenic compositionfurther comprises at least two allergens. In another embodiment, theallergen is a microbial antigen. In certain embodiments, the ratio ofthe weight of Proteosomes and liposaccharide of the immunogeniccomposition to the weight of the allergen of the immunogenic compositionis within a range from 4:1 to 1:4, within a range from 1:1 to 1:500, orwithin a range from 1:1 to 1:200. In certain particular embodiments, theallergen of the immunogenic composition is recombinant, and in otherembodiments, the allergen is a bacterial antigen. In still anotherembodiment, the allergen of the immunogenic composition is selected fromat least one of an inhaled particle, pollen, vapor, gas, food, beverage,drug, toxin, microbial antigen, dander, animal-derived compounds, dustmite feces, polypeptide, carbohydrate, and nucleic acid. In a particularembodiment, the allergen is birch pollen. In another embodiment, theimmunogenic composition is administered about one to about seven days orabout one to about 10 days after the immunomodulatory composition. Inanother embodiment, the allergic reaction is at least one of asthma,allergic alveolitis, allergic bronchopulmonary aspergillosis, allergicconjunctivitis, allergic coryza, allergic dermatitis, allergicvasculitis, and allergic rhinitis. In a particular embodiment, at leastone of the immunomodulatory composition and the immunogenic compositionfurther comprises a pharmaceutically acceptable carrier.

Also provided herein is a method for treating or preventing a microbialinfection comprising administering to a subject an immunostimulatorycomposition in an amount sufficient to elicit an innate immune response,wherein the immunostimulatory composition comprises Proteosomes andliposaccharide, and wherein the microbial infection is a viral,bacterial, parasitic, or fungal infection. In a particular embodiment,the microbial infection is a bacterial infection, wherein the bacterialinfection is a Chlamydia trachomatis infection. In another embodiment,the microbial infection is a viral infection, wherein the viralinfection is an influenza infection. In certain embodiments, theimmunostimulatory composition is administered by a route selected fromat least one of mucosal, enteral, parenteral, transdermal, transmucosal,nasal, and inhalation. In one embodiment, the liposaccharide finalcontent by weight as a percentage of Proteosome protein ranges fromabout 1% to 500%. In other embodiments, the Proteosomes andliposaccharide are obtained from the same Gram-negative bacterialspecies, and in another embodiment, the Proteosomes are obtained from afirst Gram-negative bacterial species and the liposaccharide is obtainedfrom a second Gram-negative bacterial species. In certain embodiments,the liposaccharide is obtained from a Gram-negative bacterium selectedfrom at least one of Shigella species, Chlamydia species, Yersiniaspecies, Pseudomonas species, Plesiomonas species, Escherichia species,Porphyromonas species, and Salmonella species. In a particularembodiment, Proteosomes are obtained from Neisseria species, and inanother particular embodiment, the Proteosomes are obtained fromNeisseria meningitidis, and the liposaccharide is obtained from Shigellaflexneri.

In one particular embodiment, is provided a method for treating orpreventing an influenza virus infection comprising administering to asubject an immunostimulatory composition in an amount sufficient toelicit an innate immune response, wherein the immunostimulatorycomposition comprises Proteosomes and liposaccharide, whereinProteosomes are obtained from Neisseria meningitidis, and theliposaccharide is obtained from Shigella flexneri.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show two methods for manufacturing Proteosome bulkmaterial (Flow Chart 1A and Flow Chart 1B, respectively).

FIG. 2 represents a scheme for the manufacture of Shigella flexneri 2aLPS (Flow Chart 2).

FIG. 3 presents a scheme for the manufacture of IVX-908 Proteosome-LPSadjuvant, which is also called Protollin™ (Flow Chart 3).

FIGS. 4A-4C show serum IgG, lung IgA, and lung IgG titers, respectively,from mice immunized twice intranasally with 50 μg, 20 μg, or 5 μg ofF1-V with Protollin (2.5, 1, or 0.25 μg) or without Protollin, orinjected intramuscularly with 20 μg F1-V adsorbed onto alum(Alhydrogel®). Half the mice were euthanized on day 35 post-primaryimmunization, and the remainder were euthanized on day 55. Titers areexpressed as the geometric mean of specific antibody concentrations(μg/ml for serum IgG; ng/ml for lung IgA and lung IgG); 95% confidencelimits are shown.

FIGS. 5A-5D show the survival mice after challenge with lethal doses ofaersolized Yersinia pestis. Mice were immunized twice with 20 μg of F1-Vintranasally with or without Protollin, or with 20 μg of F1-Vintramuscularly adsorbed onto Alhydrogel®, and then were challenged bywhole body exposure to 169 LD₅₀ of Y. pestis 35 days (FIG. 5A) or 55days (FIG. 5B) post-primary immunization. In a second study, miceimmunized with 50 μg of F1-V intranasally with or without 1 μg ofProtollin, or intramuscularly adsorbed onto Alhydrogel®, were challengedby whole body exposure to 254 LD₅₀ of Y. pestis 55 days post primaryimmunization (FIG. 5C). FIG. 5D shows survival of mice against challengeon day 35 or day 55 with 169 LD₅₀ of aerosolized Y. pestis. Mice wereimmunized twice with 5 μg of F1-V intranasally with Protollin at 2.5 μg,1 μg, or 0.25 μg or without Protollin. In all studies, animals in theControl group received only Protollin and died when challenged with Y.pestis.

FIG. 6A shows serum IgG levels and FIG. 6B shows lung IgA levels in miceimmunized nasally on days 0 and 14 with 5 or 25 μg of recombinantProtective Antigen (rPA) from Bacillus anthracis admixed with Protollin(1 μg) or without Protollin.

FIGS. 7A and 7B illustrate neutralization of PA-mediated killing ofmacrophages by serum and lung lavage fluid, respectively, obtained frommice that were immunized with rPA admixed with Protollin (PA+Protollin);rPA alone (PA); or rPA administered intramuscularly (PA (IM)) (figurelegend in FIG. 7B defines symbols used in both FIG. 7A and 7B).

FIG. 8A shows mortality and FIG. 8B illustrates morbidity (percentweight change) of mice that were immunized with Protollin 1 day (d-1), 2days (d-2), or 3 days (d-3) prior to challenge by inhalationadministration of mouse-adapted A/H3 influenza virus. In FIG. 8B,IVX=Protollin.

DETAILED DESCRIPTION OF THE INVENTION

Protollin™ is an outer membrane (OM)-liposaccharide (LPS) adjuvant thatincludes an outer membrane protein preparation called a Proteosome(s)(also referred to as Projuvant) prepared from Gram-negative bacteria,such as Neisseria meningitidis, and one or more liposaccharides. Asdescribed herein, Protollin may be used to elicit a potent innate immuneresponse that provides protection against pathogenic organisms.Therefore, the instant invention relates generally to the surprisingdiscovery that an immunostimulatory composition comprising Protollin canstimulate a broad spectrum, antigen-independent, nonspecific immuneresponse that can protect against a wide variety of infectious agents,including bacteria, viruses, fungi, and protozoa. In addition, Protollinmay be used to modulate or alter a detrimental immune responseminimizing damage from an overly robust inflammatory response. Hence,the instant description also pertains to the unexpected finding that animmunomodulatory composition comprising Protollin can be used tosuppress an inflammatory response, such as airway hyperresponsiveness(AHR), or to alter an immune response, thus minimizing a damaginginflammatory response (e.g., shifting a Th2 response toward a Th1phenotype). Described in more detail herein are immunostimulatory andimmunomodulatory compositions comprising Proteosome:LPS or Proteosomes,as well as immunogenic compositions comprising Proteosome:LPS orProteosomes formulated with one or more microbial antigens. In certainembodiments, the compositions are suitable for therapeutic uses such astreating or preventing a microbial infection by inducing a specificimmune response, a nonspecific immune response, or both types ofresponses. In other embodiments, the compositions described herein aresuitable for treating or preventing an inflammatory immune response,such as allergic asthma or associated complications such as AHR. Theinstant description also provides methods for preparing any of thecompositions described herein.

A Proteosome or Projuvant refers to a preparation of outer membraneproteins (OMPs, also known as porins) from Gram-negative bacteria, suchas Neisseria species (see, e.g., Lowell et al., J. Exp. Med. 167:658,1988; Lowell et al., Science 240:800, 1988; Lynch et al., Biophys. J.45:104, 1984; Lowell, in “New Generation Vaccines” 2nd ed., MarcelDekker, Inc., New York, Basil, Hong Kong, page 193 (1997); U.S. Pat. No.5,726,292; U.S. Pat. No. 4,707,543), which is useful as a carrier or anadjuvant for immunogens, such as one or more bacterial or viralantigens. Proteosomes are hydrophobic and comparable in size to certainviruses and are safe for human use. Proteosomes have the capability toauto-assemble into vesicle or vesicle-like OMP clusters of 20-800 nm,and to noncovalently incorporate, coordinate, associate, or interact(e.g., electrostatically or hydrophobically), or otherwise cooperatewith protein antigens (Ags), particularly antigens that have ahydrophobic moiety. A Proteosome includes the product of any preparationmethod that provides an outer membrane protein component in vesicular orvesicle-like form, including multi-molecular membranous structures ormolten globular-like OMP compositions of one or more OMPs. Proteosomesmay be prepared readily as described herein (see flowcharts of FIGS. 1Aand 1B) and in the art (see, e.g., U.S. Pat. Nos. 5,726,292 or5,985,284).

Liposaccharide refers to native (isolated from an organism or preparedsynthetically with a native structure) or modified lipopolysaccharide orlipooligosaccharide (collectively, also referred to as “LPS”) derivedfrom Gram-negative bacteria. For example, a liposaccharide may beisolated from or synthetically produced to have the same carbohydratestructure as a liposaccharide from Shigella flexneri or Plesiomonasshigelloides, or other Gram-negative bacteria (including species fromthe genera Alcaligenes, Bacteroides, Bordetella, Borrellia, Brucella,Campylobacter, Chlamydia, Citrobacter, Edwardsiella, Ehrlicha,Enterobacter, Escherichia, Francisella, Fusobacterium, Gardnerella,Hemophilus, Helicobacter, Klebsiella, Legionella, Leptospira (includingLeptospira interrogans), Moraxella, Morganella, Neisseria, Pasteurella,Proteus, Providencia, other Plesiomonas, Porphyromonas (includingPorphyromonas gingivalis), Prevotella, Pseudomonas, Rickettsia,Salmonella, Serratia, other Shigella, Spirillum, Veillonella, Vibrio, orYersinia species). The liposaccharide may be in a detoxified form (i.e.,having the Lipid A or Lipid A-core removed) or may be in a form that hasnot been detoxified. In the instant disclosure, the liposaccharide neednot be and preferably is not detoxified. For example, an LPS thatcontains multiple lipid A species such as P. gingivalis LPS may be usedin the compositions described herein (see, e.g., Darveau et al., Infect.Immun. 72:5041-51 (2004)). The liposaccharide may be prepared, forexample, as described in the flowchart of FIG. 2.

A Proteosome:LPS mixture or Protollin™ (also known as IVX or IVX908)described herein is a preparation of Proteosomes (Projuvant) admixed asdescribed herein with at least one kind of liposaccharide to provide anOMP-LPS composition, which can be used as an immunostimulatorycomposition. Thus, the OMP-LPS adjuvant or Protollin includes an outermembrane protein preparation of Proteosomes prepared from Gram-negativebacteria, such as Neisseria sp., (e.g., Neisseria meningitidis), and apreparation of one or more liposaccharides. Protollin may also includeone or more lipids, glycolipids, glycoproteins, small molecules, or thelike. Protollin may be prepared, for example, as described in theflowchart of FIG. 3 (see also, e.g., U.S. Patent Application PublicationNo. 2003/0044425).

Projuvant is generally used in conjunction with antigens (natural,isolated antigens or modified antigens) that possess a hydrophobicmoiety (also referred to as a hydrophobic foot). Protollin (withexogenously added LPS) can be associated with an antigen containing ahydrophobic foot or can be used with an antigen(s) that that ishydrophilic and does not contain a hydrophobic foot domain.

The present description generally provides immunostimulatorycompositions that may include a Proteosome further formulated with aliposaccharide (Protollin). For example, a Protollin composition may beused to stimulate an antigen-independent, nonspecific protective immuneresponse. In addition, the immunostimulatory composition may be used incombination with an immunogenic composition to initially promote (i.e.,stimulate, elicit, or enhance) a nonspecific immune response andsubsequently or concomitantly stimulate or elicit an adaptive immuneresponse.

By way of background and not wishing to be bound by theory, the immunesystem is designed to detect and eliminate invading pathogens bydiscriminating between self and non-self. In mammals, the immune systemis believed to have two branches; one is referred to as innate immunityand the other as adaptive immunity. The induction of innate immuneresponses may contribute significantly to overall immune defense(Medzhitov and Janeway, Trends in Microbiol. 8:452, 2000). Innateimmunity may provide a nonspecific, first line of defense to limitinfection immediately after exposure and also “network” with theadaptive immune response system by stimulating clonal responses(Hoffmann et al., Science 284:1313, 1999). Thus, a nonspecific or innateimmune response refers to an antigen-independent or antibody-independentimmune response to pathogen-associated molecular patterns (PAMPs) (see,e.g., Medzhitov and Janeway, supra), such as the specific effectsmediated by a mammalian innate immune system. For example, interactionof PAMPs with Toll-like receptors (TLRs) that are present on phagocyticantigen presenting cells (APCs) induces the release of pro-inflammatorycytokines (e.g., IFN-γ, TNF-α, and IL-12) and the up-regulation ofco-stimulatory molecules, which in turn can stimulate adaptive immunity.

An immunostimulatory composition as described herein may be any one ormore of a protein, peptide, carbohydrate, lipid, nucleic acid, chemical,or other molecule, or composition thereof, that is capable of priming,potentiating, activating, stimulating, augmenting, boosting, amplifying,or enhancing an innate immune response. An immunostimulatory agent orcomposition can mitigate, alter, treat, or prevent (e.g., as aprophylactic agent) an infectious disease or condition. A potentiated oractivated nonspecific immune response should be understood to beprotective, even providing a broad-spectrum of protection in the absenceof, or prior to, or concomitant with a specific antigen-dependent,antibody-dependent immune response. That is, an activated immuneresponse can provide protection to a host from infection by a variety ofmicroorganisms, including bacteria, viruses, parasites, or fungi.Representative examples of immunostimulatory agents or compositions asdescribed herein in more detail, include, for example, adjuvants such asProteosomes (“Projuvant”) or Protollin (Proteosomes:liposaccharrides).

Not wishing to be bound by theory, induction of an immune responsemediated by the innate immune system involves Pathogen-AssociatedMolecular Patterns (PAMPs) that may exert non-antigen, yet specificeffects. Interaction of PAMPs with Toll-Like Receptors (TLRs, at leastten of which are know and are referred to as TLR-1, TLR-2, etc.), whichare present on the cell surface of phagocytic antigen presenting cells(APCs), for example, initiate an intracellular signal transductionpathway, which in turn induces the release of pro-inflammatory cytokines(e.g., IFN-γ, TNF-α, and IL-12) and upregulation of co-stimulatorymolecules that in turn can stimulate adaptive immunity. Components ofinnate immunity recognize structures that are characteristic ofmicrobial pathogens but that are not present on mammalian cells, whichinclude unique nucleic acid structures (such as CpG DNA sequences),complex carbohydrates (such as LPS), as well as bacterial proteins,lipoproteins, and peptidoglycans. For example, Neisserial porin proteins(e.g., porin A, porin B, which are used to prepare Proteosomes) arerecognized by TLR-2, and LPS from Gram-negative bacteria (which is acomponent of Protollin) is recognized by TLR-4. The Proteosome(Projuvant) and Protollin adjuvants may be used to stimulate an innateimmune response. Moreover, through engagement of two components ofProtollin (protein and liposaccharide) with TLRs on APCs, Protollin mayinitiate a chain of events that leads to the induction of both innateand adaptive immunity. In addition to Toll-like receptor activation ofinnate immunity, Protollin may activate other immune system componentsor immune functions. LPS is understood to be immunostimulatory throughinteractions with TLR-4 receptors present on the cell surface of certainimmune system cells; hence, an immune response stimulated or elicited byProteosomes (Projuvant) and an immune response stimulated or elicited byProtollin may be qualitatively or quantitatively distinguished in astatistically significant manner that correlates with the ratio of OMPto LPS in Protollin. The Protollin compositions described herein mayalso include an LPS that may interact with more than one Toll-likereceptor such as the LPS obtained from Porphyromonas gingivalis (see,e.g., Darveau et al., Infect. Immun. 72:5041-51 (2004)).

An adaptive immune response (i.e., specific or acquired) includesresistance to an infectious agent or an antigen that is mediated by theimmune system and that results from previous exposure to the infectiousagent or antigen. For example, specific immunity can be a result of anaturally acquired (patent or latent) infection or from an intentionalvaccination. In addition, specific immunity may be passively andtransitorily acquired from the natural transfer of antibodies fromanother (e.g., maternally inherited), or from exogenous transfer ofantibodies or immune cells by intentional inoculation (sometimesreferred to as passive immunotherapy).

An immunogenic composition as described herein comprises one or morecompounds, antigens, immunogens, or agents capable of priming,eliciting, potentiating, activating, stimulating, augmenting, boosting,amplifying, or enhancing an adaptive (specific) immune response, whichmay be cellular (T cell) or humoral (B cell), or a combination of a Tcell and a B cell response. Preferably, the adaptive immune responsewill be protective. A representative example of an immunogen is amicrobial antigen, such as one or more bacterial, viral, fungal, orparasitic proteins of interest.

An immunomodulatory composition as described herein may compriseProteosomes or Protollin adjuvants and any one or more of a protein,peptide, chemical, or other molecule, or composition thereof, that iscapable of altering (modifying, modulating, adjusting, regulating) (orincreasing (potentiating) or decreasing (suppressing) in a statisticallysignificant manner or in a clinically significant manner) one or moreimmune functions. An immunomodulatory agent or composition can mitigate,ameliorate, treat, or prevent (e.g., as a prophylactic agent) anundesired or abnormal inflammatory response. An immune function caninclude a cellular response with a particular pattern of cytokineproduction (e.g., Th1, Th2), a humoral response (e.g., antibodyproduction), or a combination thereof, to a particular microbe orantigen. For example, if a subject previously exposed to an allergen(i.e., is sensitized) comes into contact with the allergen again,allergic asthma may develop due to a Th2 response characterized by anincreased production of type 2 cytokines (IL-4, IL-5, IL-9, IL-13)secreted by CD4+ T lymphocytes. An immunomodulatory composition asdescribed herein may alter the Th2 response by, for example, shiftingthe response toward a Th1 phenotype that is less damaging to the airway.That is, an altered (or modulated) immune response can provideprotection to a host against infection by a variety of microorganisms(including bacteria, viruses, parasites, or fungi) or againstinflammatory responses (e.g., allergy, asthma, nasal polyps) caused byantigens.

An allergic reaction as described herein refers to a local or generalreaction in a subject following contact with a specific antigen(allergen) to which the subject had been previously exposed andsensitized. The immunologic interaction of endogenous or exogenousantigen with antibody or sensitized lymphocytes can give rise toinflammation and tissue damage—in other words, allergy is an immunereaction resulting in damage to self-tissues and cells, usually throughinflammatory reactions. Extrinsic or allergic asthma (also referred toherein as reactive airway disease) is an inflammatory disease of thelungs characterized by a generally reversible airway obstruction.Features of allergic asthma include elevated concentrations of serumIgE, pulmonary eosinophilia, airway hyperresponsiveness, excessiveairway mucus production, and airway remodeling marked by peribronchiolarcollagen deposition and increases in airway smooth muscle mass. Otherexemplary allergic reactions or inflammatory conditions include allergicalveolitis, allergic bronchopulmonary aspergillosis, allergicdermatitis, eczema, allergic conjunctivitis, allergic coryza, allergicvasculitis, rhinosinusitis, and allergic rhinitis.

Hyperresponsiveness relates to an abnormal response or condition inwhich a foreign agent elicits an exaggerated immune response. Forexample, allergic asthma may be a result of repeated exposure toairborne allergens that trigger detrimental immunological responses,such as persistent inflammation in the bronchial wall, which can resultin structural and functional changes in the respiratory system. Afterallergen inhalation by sensitized subjects (i.e., those subjects thathave already been exposed to the allergen), the immune response isdependent on CD4+ T lymphocytes that are skewed to a T helper (Th) 2phenotype. Th2 cytokines, for example, IL-4, IL-5, IL-9, and IL-13 areimportant to asthma pathogenesis. For example, IL-4 drives the T helperresponse in favor of Th2, resulting in enhanced production of IgE; IL-5,which with granulocyte macrophage colony stimulating factor (GM-CSF) andIL-3, is important for the production of eosinophils; and IL-13, whichis required for airway hyperresponsiveness and mucous metaplasia, whichare downstream pathophysiological features that are closely linked withclinical asthma. All these cytokines, together with TGF-beta have beenimplicated in airway remodeling. While the role of eosinophils in thepathology of asthma is not entirely understood, the number of airwayeosinophils is associated directly with disease severity (see, e.g., Leeet al., Science 305:1773 (2004); Humbles et al., Science 305:1776(2004)). The resulting structural and morphometric changes (remodeling)include subepithelial fibrosis, goblet cell hyperplasia and metaplasia,which result in functional consequences such as loss of distensibilityof asthmatic airways, bronchial hyperreactivity (even in the absence ofthe allergen), and an accelerated progressive decrease in forcedexpiratory volume at 1 second time intervals (FEV₁). The Th2 cytokinesmay also prime and activate eosinophils to release proinflammatoryagents, lipid mediators, and other cytokines thought to contribute tothe observed tissue damage, remodeling, and hyperresponsiveness.

Tolerance as used herein refers to the ability to endure or be lessresponsive to a stimulus, especially over of a period of continuedexposure, such as to an allergen. For example, immunologic tolerancerefers to a natural or artificially induced state of reduced ornon-responsiveness to a specific antigen or allergen.

In the present description, any concentration range, percentage range,ratio range or other integer range is to be understood to include thevalue of any integer within the recited range and, when appropriate,fractions thereof (such as one tenth and one hundredth of an integer),unless otherwise indicated. As used herein, “about” or “comprisingessentially of” mean±15%. The use of the alternative (e.g., “or”) shouldbe understood to mean one, both, or any combination thereof of thealternatives. As used herein, the use of an indefinite article, such as“a” or “an,” should be understood to refer to the singular and theplural of a noun or noun phrase. In addition, it should be understoodthat the individual compositions, formulations, or compounds, or groupsof compositions, formulations, or compounds, derived from the variouscomponents or combinations of the composition or sequences, structures,and substituents described herein, are disclosed by the presentapplication to the same extent as if each composition or compound orgroup of compositions or compounds was set forth individually. Thus,selection of particular sequences, structures, or substituents is withinthe scope of the present invention.

In one embodiment, an immunomodulatory composition may comprise aProteosome formulated with a liposaccharide, that is, Protollin. Forexample, a Protollin composition can be used to suppress or inhibit anundesired immune response or to induce or promote tolerance to anundesired immune challenge (e.g., shift a Th2 cytokine productionphenotype to a Th1 phenotype). In addition, the immunomodulatorycompositions described herein can be used in combination with animmunogenic composition to initially promote suppression of an undesiredimmune response, and subsequently or concomitantly, promote induction oftolerance. By way of background and not wishing to be bound by theory, Tlymphocytes, in particular CD4⁺ T cells that produce Th2 cytokines andthat have undergone an aberrant expansion, play an important role in thepathogenesis of asthma. In a murine model, the administration of agentssuch as IL-12 and IFN-γ or CpG oligodeoxynucleotides can inhibit Th2cytokine production and stimulate Th1 lymphocytes and/or cytokines toprevent the development of antigen-induced airway hyperresponsiveness(AHR) and inflammation (see Lack et al., J. Immunol. 157:1432 (1996);Gavett et al., J. Exp. Med. 182:1527-36 (1995); Kline et al., J.Immunol. 160:2555 (1998)).

In certain embodiments, the immunostimulatory compositions describedherein are useful for eliciting a nonspecific (or innate) immuneresponse. Such an immunostimulatory composition may provide anonspecific protective response that prevents or treats a microbialinfection in a host or subject. The immunostimulatory compositiondescribed herein may also be used to stimulate an innate (nonspecific)immune response that potentiates or enhances an adaptive immune responseelicited by subsequently administered vaccine, for example, animmunogenic composition comprising Protollin formulated with a microbialantigen, such as F1-V plague antigen, Protective Antigen from Bacillusanthracis, or a bacterial antigen from Chlamydia trachomatis,enteropathogenic E. coli, or another pathogenic bacteria.

In certain embodiments, immunostimulatory and immunogenic compositionsmay be administered simultaneously to elicit an innate immune responsewhile at the same time potentiating or priming an adaptive immuneresponse. In certain other embodiments, immunomodulatory and immunogeniccompositions may be administered simultaneously to elicit an alteredimmune response while at the same time potentiating or primingtolerance. Alternatively, short-term use of an immunostimulatory orimmunomodulatory composition as described herein may be used withoutsubsequent or simultaneous treatment with an immunogenic composition.Nonspecific protection or an altered immune response (without subsequentor simultaneous immunogenic composition treatment) may last from about 1day to 3 months or longer. For example, animals remained protected fromChlamydia challenge at least 11 weeks after treatment withProteosome-LPS (Protollin) (see Example 15).

In other embodiments, the immunomodulatory compositions described hereinare useful for altering an inflammatory immune response. As set forthherein, the current compositions may be used to alter an inflammatoryimmune response (e.g., cause a shift from a Th2 to a Th1 phenotype) thatmay potentiate or enhance the development of tolerance to a specificantigen.

An immunostimulatory or immunomodulatory composition comprises anadjuvant, preferably a Proteosome or a Proteosome:LPS adjuvant.Proteosomes can be comprised of outer membrane proteins (OMPs or porins)from Neisseria species, but can also be derived from other Gram-negativebacteria (see, e.g., Lowell et al., J. Exp. Med. 167:658, 1988; Lowellet al., Science 240:800, 1988; Lynch et al., Biophys. J. 45:104, 1984;U.S. Pat. No. 5,726,292; U.S. Pat. No. 4,707,543), or a combination ofNeisseria OMPs and OMPs from at least one other Gram-negative bacteria.By way of background and not wishing to be bound by theory, mixing ofProteosomes with a protein (e.g., a microbial antigen) provides acomposition comprising non-covalent association, interaction, orcoordination between the microbial antigen and Proteosomes, whichassociation or coordination forms when the detergent used to solubilizethe Proteosomes is selectively removed or reduced, for example, bydialysis or diafiltration.

Proteosomes may be used as an adjuvant (i.e., a component of animmunostimulatory or immunomodulatory composition) and/or may be used asan antigen delivery composition (i.e., an immunogenic composition). Inone embodiment, an immunogenic composition comprises one or moremicrobial antigens (i.e., bacterial, parasitic, fungal, or viralantigens or immunogens, or variants and fragments thereof) and anadjuvant, wherein the adjuvant comprises Projuvant (i.e., Proteosome) orProtollin (i.e., Proteosome:LPS). A preferred microbial antigen is onethat stimulates or elicits an immune response (either humor orcell-mediated) that protects (prevents a microbial infection, reducesthe microbial load, kills the microorganism or prevents its propagation)the host or subject.

In certain embodiments, the immunostimulatory or immunomodulatorycomposition may be a Proteosome further formulated with aliposaccharide. That is, the Proteosome adjuvant (Projuvant) may beprepared to include an additional (e.g., exogenous or endogenous)immunostimulatory or immunomodulatory molecule, such as LPS.Liposaccharride can be prepared synthetically, isolated from abiological source (e.g., non-detoxified), chemically modified (e.g.,detoxified or otherwise chemically modified by adding, deleting, orchanging substituents), or any combination thereof, For example, theProjuvant may be admixed as described herein with liposaccharide toprovide an OMP:LPS adjuvant (i.e., Protollin). These two components ofProtollin may be formulated at specific initial ratios (see flowchart ofFIG. 3) to optimize their interaction, resulting in stable associationand formulation of the components for use in an immunostimulatory orimmunomodulatory composition. The process for making Protollin generallyinvolves mixing the components in a selected detergent solution (e.g.,Empigen® BB (n-Dodecyl-N,N-dimethylglycine), Triton® X-100 (octyl phenolethoxylate), or Mega-10 (n-Decanoyl-N-methylglucamide)) or otherdetergent (e.g., octoglucoside). Complex formation of the OMP and LPScomponents occurs while reducing the amount of detergent to apredetermined, preferred concentration, by dialysis or bydiafiltration/ultrafiltration methodologies. The duration of dialysiscan be adjusted to retain varying amounts of detergent in the vaccineformulation including, for example, concentrations from 250, 500, 750,1000 ppm, or more, or even lower amounts (e.g., 50 ppm). Mixing,co-precipitation, or lyophilization of the two components may also beused to effect an adequate and stable association or formulation. Incertain embodiments, the Protollin may be formulated to comprise LPSfrom one bacteria or may be formulated to comprise two or moreliposaccharides obtained from different bacteria. For example, oneProtollin formulation may include liposaccharide from Escherichia andShigella, or from Chlamydia and Yersinia, or Phorphyromonas andShigella, or from Neisseria, Escherichia, Yersinia, and Shigella, and soon. A Protollin formulation may be optimized with one or a plurality ofas many different liposaccharides as is necessary or desired.

Protollin compositions described herein may contain liposaccharidederived from any Gram-negative bacterial species, which may be the sameGram-negative bacterial species that is the source of Proteosomes, ormay be a different bacterial species. In one embodiment, the finalliposaccharide content by weight as a percentage of the total Proteosomeprotein may be in a range from about 0.1% to about 10%, from about 0.5%to about 5%, from about 1% to about 500%, or in a range from about 10%to about 100%, about 5% to about 20% or from about 10% to about 50%, orin a range from about 20% to about 200%, or in a range from about 30% toabout 150% or from about 50% to 150%. In a preferred embodiment, theimmunostimulatory composition comprises a Proteosome component preparedfrom Neisseria meningitidis and the liposaccharide prepared fromShigella flexneri or Plesiomonas shigelloides, such that the finalliposaccharide content is between 50% to 150% of the total Proteosomeprotein by weight. In another embodiment, Proteosomes are prepared withendogenous lipooligosaccharide (LOS) content from Neisseria ranging fromabout 0.5% up to about 5% of total OMP. In another embodimentProteosomes are provided that comprise endogenous liposaccharide (i.e.,from the same bacteria as the Proteosomes) in a range from about 12% toabout 25%, and in a preferred embodiment between about 15% and about 20%of total OMP. Alternatively, mutant bacteria that can no longer produceLPS (e.g., a Neisseria LPS—minus strain) can be used to prepareProjuvant such that the OMP:LPS mixture has 0% endogenous LPS.Accordingly, Protollin may have exogenous LPS, endogenous LPS, or acombination thereof, wherein the exogenous and endogenous LPS may bepresent in equal amounts or at different ratios.

The present invention is also directed generally to the use of microbialantigens in combination with an immunostimulatory or immunomodulatorycomposition to generate an immunogenic composition. The antigens arepreferably from clinically relevant microorganisms, such as bacteria,including pathogenic bacteria; viruses (e.g., Influenza, Measles,Coronavirus); parasites (e.g., Trypanosome, Plasmodium, Leishmania);fungi (e.g., Aspergillus, Candida, Coccidioides, Cryptococcus); and thelike. For example, the antigen may be from bacteria, particularlypathogenic bacteria, such as the causative agent of anthrax (Bacillusanthracis), plague (Yersinia pestis), stomach cancer (Helicobacterpylori), sexually transmitted diseases (Chlamydia trachomatis orNeisseria gonorrhea), and the like. Other representative examplesinclude antigens from certain viruses, such as influenza virus(es),Norwalk virus, smallpox virus, West Nile virus, SARS virus, respiratorysyncytial virus, measles virus, and the like. Exemplary fungi includeCandida albicans or Aspergillus spp., and exemplary parasites includethe causative agents of trypanosomiasis, leishmania, pneumonic plague,and lyme disease (Borrellia burgdorferi).

As described herein, the antigens can be prepared recombinantly,synthetically, isolated from a biological source, recombinantly orchemically modified, and any combination thereof. A biological sourceincludes but is not limited to a biological sample from a host orsubject (e.g., tissue, blood, serum, plasma, lung lavage, nasal wash),bacterial cell culture, or tissue cell culture. A “sample” as usedherein refers to a biological sample and may be provided by obtaining ablood sample, biopsy specimen, tissue explant, organ culture, or anyother tissue or cell preparation from a subject or a biological source.A sample may further refer to a tissue or cell preparation in which themorphological integrity or physical state has been disrupted, forexample, by dissection, dissociation, solubilization, fractionation,homogenization, biochemical or chemical extraction, pulverization,lyophilization, sonication or any other means for processing a samplederived from a subject or biological source.

A microbial antigen or fragment thereof can be prepared from a varietyof biological sources, such as tissues of an infected subject orcultured cell lines. Primary isolation may be from, for example,peripheral blood cells or from respiratory secretions or excretions.Preferably, the isolated microbes are propagated or cultured onappropriate culture media that are known to skilled artisans, in primarycell cultures, or on established cell lines known in the art as requiredfor a particular microbe. In certain embodiments, the antigens orfragments thereof are isolated from intact microbial particles. As usedherein, the term “isolated” or “derived from” means that the material isremoved from its original or natural environment. For example, anaturally occurring nucleic acid molecule or polypeptide present in aliving animal or cell or virus is not isolated, but the same nucleicacid molecule or polypeptide is isolated when separated from some or allof the co-existing materials in the natural system. An isolated nucleicacid molecule or a nucleic acid molecule that is removed from itsnatural environment includes a vector such as a recombinant expressionvector, which comprises a nucleic acid molecule that encodes a microbialantigen. In other embodiments, peptides or polypeptides, such asantigens or variants and fragments thereof, may be either partiallypurified or purified to homogeneity.

Also provided herein are methods for producing synthetic microbialantigens, including fusion proteins that comprise a microbial antigen,variant, or fragment thereof A peptide or polypeptide component of animmunogenic composition may be synthesized by standard chemical methods,including synthesis by an automated procedure. In general, immunogenicpolypeptides or peptides are synthesized based on the standardsolid-phase Fmoc protection strategy with HATU as the coupling agent.The immunogenic peptide can be cleaved from the solid-phase resin withtrifluoroacetic acid containing appropriate scavengers, which alsodeprotects side chain functional groups. Crude immunogenic peptide maybe further purified using preparative reverse phase chromatography.Other purification methods, such as partition chromatography, gelfiltration, gel electrophoresis, or ion-exchange chromatography may beused. Other synthesis techniques known in the art may be employed toproduce similar immunogenic peptides, such as the tBoc protectionstrategy, use of different coupling reagents, and the like. In addition,any naturally or non-naturally occurring amino acid or derivativethereof may be used, including D- or L-amino acids and combinationsthereof.

As described herein, the microbial antigens or fragments thereof of theinvention may be recombinant, wherein a recombinant nucleic acidexpression construct comprises a polynucleotide that encodes the antigenand is operatively linked to an expression control sequence (e.g.,promoter, enhancer). Recombinant polynucleotide expression constructsmay be prepared according to methods known to persons skilled in themolecular biology art. Cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described, for example, in Sambrooket al., Molecular Cloning: A Laboratory Manual, Third Edition, ColdSpring Harbor, N.Y., (2001), and may include plasmids, cosmids, shuttlevectors, viral vectors, and vectors comprising a chromosomal origin ofreplication as disclosed therein. Recombinant expression constructs alsocomprise expression control sequences (regulatory sequences) that allowexpression of a polypeptide of interest in a host cell, including one ormore promoter sequences (e.g., lac, tac, trc, ara, trp, λ phage, T7phage, T5 phage promoter, CMV, immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I),enhancer sequences, operator sequences (e.g., lacO), and the like.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences. In preferred embodiments theconstructs are included in compositions that are administered in vivo.Such vectors and constructs include chromosomal; nonchromosomal; andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA; viral DNA, such as vaccinia, adenovirus, fowl pox virus,and pseudorabies; or replication deficient retroviruses as describedbelow. However, any other vector may be used for preparation of arecombinant expression construct, and in preferred embodiments such avector will be replicable and viable in the host (subject).

The recombinant expression vector may be introduced into a host cell bytransformation, transfection, or transduction according to methods knownto those skilled in the molecular biology art. The host cells (such as aeukaryotic or prokaryotic host cells or insect cells) may be cultured topermit expression of the encoded microbial antigen, thus producing arecombinant protein antigen (or immunogen), or fragment thereof. Theantigens may be further fused or conjugated to another amino acidsequence, which sequence may be a hydrophobic anchor or foot (anch) tofacilitate or otherwise enhance non-covalent association with Projuvantor Protollin. A fragment of a microbial antigen polypeptide may compriseany portion of such a polypeptide that has at least one epitope capableof eliciting a protective immune response (cellular or humoral) againsta microbial infection. Immunogenic polypeptides may also be arranged orcombined and linked in a linear form, and each immunogen may or may notbe reiterated, wherein the reiteration may occur once or multiple times.In addition, a plurality of different immunogenic polypeptides (e.g.,protein variants, or fragments thereof) can be selected and mixed orcombined into a cocktail composition to provide a multivalent vaccinefor use in eliciting a protective immune response.

A variant of an antigen, including a microbial antigen or allergen asdescribed herein, or a fragment of an antigen or variant, includemolecules that are structurally similar and functionally similar. Avariant or fragment of antigen or allergen, is functionally similar tothe antigen or allergen if the variant or fragment is capable ofeliciting an immune response at least comparable according to one ormore characteristics or parameters of an immune response to thatelicited by the antigen or allergen, which may be determined usingmethods, including animal models and in vitro assays, described hereinand practiced in the art. For example, a comparable immune response maybe determined by quantitative and/or qualitative determination ofcytokine production, antibody production (including class and/orisotype), and protection as determined in an animal model. A comparableimmune response of an antigen variant or fragment to the antigen may beindicated by statistical analysis of a particular measure (such ascytokine production or immunoglobulin production) and maybe within 5%,10%, 15%, or 20% or 25% of the measurement. A functionally similarvariant or fragment also is capable of binding to an antibody thatspecifically binds to the antigen or allergen.

Such variants include naturally-occurring polymorphisms or allelicvariants, microbial strain variants, as well as synthetic polypeptides(or the polynucleotides encoding the variant polypeptides) that containconservative amino acid substitutions of the amino acid sequences. Avariety of criteria known to those skilled in the art indicate whetheramino acids at a particular position in a peptide or polypeptide aresimilar. For example, a similar amino acid or a conservative amino acidsubstitution is one in which an amino acid residue is replaced with anamino acid residue having a similar side chain, which include aminoacids with basic side chains (e.g., lysine, arginine, histidine); acidicside chains (e.g., aspartic acid, glutamic acid); uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine, histidine); nonpolar side chains (e.g., alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan); beta-branched side chains (e.g., threonine, valine,isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan). Proline, which is considered more difficult to classify,shares properties with amino acids that have aliphatic side chains(e.g., Leu, Val, Ile, and Ala). In certain circumstances, substitutionof glutamine for glutamic acid or asparagine for aspartic acid may beconsidered a similar substitution in that glutamine and asparagine areamide derivatives of glutamic acid and aspartic acid, respectively.

Variant polynucleotides and their encoded polypeptide products can beidentified by determining whether the polynucleotides hybridize with anucleic acid molecule having the nucleotide sequence of under highlystringent or moderately stringent conditions. As an alternative, variantpolynucleotides and the encoded polypeptides can be identified bysequence comparison. As used herein, two amino acid sequences have “100%amino acid sequence identity” if the amino acid residues of the twoamino acid sequences are the same when aligned for maximalcorrespondence. Similarly, two nucleotide sequences have “100%nucleotide sequence identity” if the nucleotide residues of the twonucleotide sequences are the same when aligned for maximalcorrespondence. Sequence comparisons can be performed using any standardsoftware program, such as BLAST, tBLAST, pBLAST, or MegAlign. Stillothers include those provided in the Lasergene bioinformatics computingsuite, which is produced by DNASTAR® (Madison, Wis.). References foralgorithms such as ALIGN or BLAST may be found in, for example,Altschul, J. Mol. Biol. 219:555-565, 1991; or Henikoff and Henikoff,Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992. BLAST is available atthe NCBI website. Other methods for comparing multiple nucleotide oramino acid sequences by determining optimal alignment are well known tothose of skill in the art (see, e.g., Peruski and Peruski, The Internetand the New Biology: Tools for Genomic and Molecular Research (ASMPress, Inc. 1997); Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997); and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition, Academic Press, Inc.,1998). An antigen or allergen and a variant thereof should have at leasta 50% amino acid sequence identity to and preferably, greater than 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% identity.

Variants may be prepared readily using mutagenesis techniques known andpracticed in the art. For example, site-directed mutagenesis (e.g.,Kramer et al. (Nucleic Acids Res. 12, 9441, (1984)); the AnglianBiotechnology Ltd handbook; Kunkel Proc. Natl. Acad. Sci. USA 82:488-92(1985); Kunkel et al., Methods in Enzymol. 154:367-82 (1987)) and randommutagenesis techniques, such as alanine scanning mutagenesis, errorprone polymerase chain reaction mutagenesis, andoligonucleotide-directed mutagenesis are well known and used extensivelyin the art (see, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3^(nd) ed., Cold Spring Harbor Laboratory Press, NY (2001)).

Methods for preparing the immunostimulatory compositions,immunomodulatory compositions, and immunogenic compositions aredescribed herein and are known in art (see, e.g., U.S. PatentApplication Publications Nos. 2001/0053368 and 2003/0044425). Theantigen(s) and adjuvant are formulated at specific initial ratios(weight:weight) to optimize interaction (or cooperation) between thecomponents resulting in non-covalent association (or nonspecificjuxtaposition) of a significant portion of the two components with eachother. For example, a mixture of at least one antigen with a Proteosome(Projuvant) or Protollin is prepared in the presence of detergent, andreduction of the concentration of the detergent or removal of thedetergent from the mixture by diafiltration/ultrafiltration leads toassociation (interaction or coordination) of the antigen(s) with theadjuvant (see FIG. 3). The ratio of Proteosome or Protollin to antigenafter the mixture has been dialyzed, diafiltered, or ultrafiltered maybe the same or may be altered (increased or decreased) from the initialratio. In certain embodiments, the initial orpost-dialysis/diafiltration/ultrafiltration Proteosome or Protollin (theweight of Protollin equals the combined weights of the Proteosomes andliposaccharide) to antigen ratio (wt/wt) in an immunogenic compositionmixture ranges from about 1:1 to about 4:1. The ratio may range from 1:1to about 8:1 or higher. In certain other embodiments, the Proteosome orProtollin to antigen ratio (wt/wt) in the mixture ranges from about 1:1to about 1:500, or in a range of about 1:1 to about 1:200 or about 1:2to about 1:200, or in a range of about 1:2 to about 1:100, or in a rangeof about 1:5 to about 1:50, or in a range of about 1:2 to about 1:20.The detergent-based solutions of the two components may contain the samedetergent or different detergents, and more than one detergent may bepresent in the mixture subjected to ultrafiltration/diafiltration.Suitable detergents include Triton®, Empigen® BB, and Mega-10. Otherdetergents can also be used (e.g., octoglucoside). The detergents serveto solubilize the components used to prepare the composition. The use ofa mixture of detergents may be particularly advantageous. Thedetergent(s) are removed or the concentration is reduced bydiafiltration/ultrafiltration prior to final formulation.

Also contemplated are methods for treating or preventing a microbialinfection, by administering an immunostimulatory composition describedherein for eliciting a nonspecific protective immune response. Inanother embodiment, a method is provided for treating or preventing amicrobial infection by administering an immunostimulatory compositionfor eliciting an innate immune response and administering an immunogeniccomposition for eliciting an adaptive immune response. Also contemplatedare methods for altering an inflammatory response, or treating orpreventing an allergic reaction, using immunomodulatory and/orimmunogenic compositions of this disclosure. An immunostimulatorycomposition, immunomodulatory composition, or immunogenic compositionmay further include a pharmaceutically acceptable vehicle, carrier,diluent, and/or excipient, in addition to one or more microbial antigens(or immunogens) or fragment or fusion thereof and, optionally, othercomponents. For example, pharmaceutically acceptable carriers or othercomponents suitable for use with immunostimulatory compositions,immunomodulatory compositions, or immunogenic compositions include athickening agent, a buffering agent, a solvent, a humectant, apreservative, a chelating agent, an additional adjuvant, and the like,and combinations thereof.

In addition, the pharmaceutical compositions as described herein mayfurther include a diluent such as water or phosphate buffered saline(PBS). In certain embodiments, the diluent is PBS with a final phosphateconcentration range from about 0.1 mM to about 1 M, from about 0.5 mM toabout 500 mM, from about 1 mM to about 50 mM, or from about 2.5 mM toabout 10 mM; and the final salt concentration ranges from about 100 mMto about 200 mM or from about 125 mM to about 175 mM. In anotherembodiment, the final PBS concentration is about 5 mM phosphate andabout 150 mM salt (such as NaCl). In certain embodiments, any of theaforementioned immunostimulatory, immunomodulatory, or immunogeniccompositions further comprising a diluent will be sterile.

The compositions can be sterilized either by preparing them under anaseptic environment or by terminal sterilization using methods availablein the art. Many pharmaceuticals are manufactured to be sterile and thiscriterion is defined by USP XXII <1211>. The term “USP” refers to U.S,Pharmacopeia (Rockville, Md.). Sterilization may be accomplished by anumber of means accepted in the industry and listed in USP XXII <1211>,including gas sterilization, ionizing radiation, or filtration.Sterilization may be maintained by what is termed aseptic processing,defined also in USP XXII <1211>. Acceptable gases used for gassterilization include ethylene oxide. Acceptable radiation types usedfor ionizing radiation methods include gamma, for instance from a cobalt60 source and electron beam. A typical dose of gamma radiation is 2.5MRad. When appropriate, filtration may be accomplished using a filterwith suitable pore size, for example, 0.22 μm and of a suitablematerial, for instance Teflon®. The preparation of Proteosomes orProtollin results in particles small enough that an immunogeniccompositions can be filtered through a 0.8 μm filter, a 0.45 μm filter,or a 0.2 μm filter. Thus, in certain embodiments the immunostimulatory,immunomodulatory, and/or immunogenic compositions of this invention canbe filter sterilized. This is highly advantageous to eliminate anycomplications due to the presence of contaminants.

In one embodiment, a method is provided for eliciting a nonspecificprotective immune response, comprising administering to a subject (orpatient) in need thereof an amount of an immunostimulatory compositionand under conditions sufficient to elicit, induce, or stimulate animmune response such that the amount of the immunostimulatorycomposition is therapeutically effective. The conditions under which animmune response is elicited in a subject include a variety of parametersand criteria described herein and understood by persons having skill inthe medical art, and include but are not limited to the time of dosing,number of doses, route of administration, and the like. A nonspecificprotective immune response as described herein includes an innate immuneresponse that is not a specific antigen-dependent or antibody-dependentresponse (that is, does not involve clonal expansion of T cells and/or Bcells) and may be elicited by any one of numerous antigens, immunogens,or microorganisms. The immunostimulatory composition comprisesProteosomes and liposaccharide (Protollin), either one of which or bothmay elicit a nonspecific protective response. When the immunostimulatorycomposition is used to elicit a nonspecific immune response or an innateimmune response for treating or preventing a microbial infection, suchas a bacterial infection or a viral infection, the immunostimulatorycomposition comprising Protollin may not contain a liposaccharide fromthe genus of bacteria that is the causative agent of an infection to betreated or prevented. That is, the Protollin need not have components orPAMPs from the organism that is causing an infection or that may causean infection. By way of example, an immunostimulatory compositioncomprising Proteosomes obtained from Neisseria meningitidis and LPSobtained from Shigella flexneri may be used to stimulate an innateresponse in a subject that provides protection, that is, treats orprevents infection caused by a virus, such as an influenza virus, or bya bacteria such as Yersinia pestis, Bacillus anthracis, or Chlamydiatrachomatis. Accordingly, the immunostimulatory compositions describedherein may be useful for treating or preventing infections that can becaused by one of numerous different strains of a virus, such asdifferent strains of influenza virus, or that may be caused by one ofnumerous different strains, serotypes, or immunotypes of a bacterialspecies.

The Proteosomes and liposaccharide of Protollin may be obtained from thesame or different bacterial genera or species. The Proteosomes may beobtained from a Gram-negative bacteria such as a Neisseria species andthe liposaccharide may be from another Gram-negative bacteria such asfrom Shigella, Chlamydia, Plesiomonas, Porphyromonas, or E. coli. In oneembodiment, a method is provided for potentiating a specific immuneresponse, comprising administering to a subject in need thereof atherapeutically effective amount of an immunostimulatory composition,wherein the immunostimulatory composition comprises Proteosomes andliposaccharide.

In another embodiment, a method is provided for treating or preventing amicrobial infection, wherein after the immunostimulatory composition hasbeen administered, an immunogenic composition is administered to thesubject (or patient) in need thereof in an amount sufficient and underconditions such that the administration of both compositions effectivelyelicits a specific immune response. In a certain embodiment, theimmunogenic composition comprises Proteosomes, liposaccharide, and anantigen such as a microbial antigen (bacterial, viral, parasitic, orfungal antigen). The immunogenic composition may comprise Proteosomesthat are obtained from the same or different sources of the Proteosomesof the immunostimulatory composition, such as different Gram-negativebacteria genus and/or species. Similarly, an LPS component of theimmunogenic composition and the immunostimulatory composition may befrom the same or different bacteria. The immunogenic composition maycomprise one antigen that is a microbial antigen, or may comprise 2, 3,4, 5, 6, 7, or 8-10 microbial antigens. When at least two microbialantigens are contained in the immunogenic composition, the antigens maybe obtained from, associated with, or known to be originally derivedfrom the same microorganism or from different microorganisms.Alternatively, the immunogenic composition may comprise at least oneantigen without Proteosomses and/or LPS, or the immunogenic compositionmay comprise at least one antigen and an adjuvant such as alum. Theantigen may be isolated (purified) or partially isolated (or purified),or may be delivered as a live, infectious microorganism or in anattenuated form. In certain embodiments, the microbial antigen is aviral split antigen as described herein, which may contain allcomponents of a virus. Any one of the immunogenic compositions describedherein may be administered to a subject once or more than once (multipletimes) after administration of an immunostimulatory composition.

The immunostimulatory and immunogenic compositions described herein maybe administered to a subject (or patient) as a prophylactic treatment toprevent a microbial infection prior to exposure to the microorganismthat causes the infection. A prophylactic treatment also includesadministration of an immunostimulatory composition alone or followed byan immunogenic composition to prevent a microbial infection in a subjectwho is known to have been exposed, who is at risk for exposure, or whohas likely been exposed to the causative microbial agent. Animmunostimulatory composition alone or followed by an immunogeniccomposition may also be used to treat a subject who may have asubclinical infection (i.e., not detected according to appropriateclinical criteria) or may have a clinical infection that is or can bediagnosed clinically according to criteria known to those skilled in theart, including symptomatology, clinical chemistry, and microbiologicalanalyses.

The ratio (wt:wt) (initial ratio or post-removal of detergent) ofProteosomes or Protollin (combined weight of Proteosomes andliposaccharide) to antigen of the immunogenic composition may range fromabout 4:1 to about 1:4, and may be at least 4:1 or at least 2:1. Theratio of Proteosomes (or Protollin) to antigen may be greater than 1:1,greater than 2:1, greater than 3:1 and greater than 4:1. The ratio canbe as high as 8:1 or higher. Alternatively, the ratio of Proteosome (orProtollin) to antigen in the mixture is 1:1, 1:2, 1:3, 1:4, or 1:8. TheProteosome or Protollin to antigen ratio in the mixture may range fromabout 1:1 to about 1:500, or from about 1:1 to about 1:200 or from about1:2 to about 1:200, or from about 1:2 to about 1:100, or from about 1:5to about 1:50, or from about 1:2 to about 1:20.

As described herein, different sources of LPS may be used in Protollinpreparations. The use of a particular source or type of LPS may dependupon the adjuvant properties of the Proteosome:LPS composition whenadministered by a particular route, such as intranasally, the type ofimmune response induced (innate and/or adaptive), the quantity orquality of cytokine production, the capability of a particular LPS typeto interact with a particular host cell, the solubility properties ofthe LPS (i.e., the length of a O-polysaccharide chain may influencesolubility of the Proteosome/LPS mixture during preparation ofProtollin), as well as production methods (e.g., yield, biohazardcontainment requirements). Protollin may be prepared containing S.flexneri 2a LPS, LPS from different strains of E. coli, or LPS fromother Gram-negative bacteria and characterized according to methodsdescribed herein and known in the art.

The ratio of Proteosome (OMPs) to LPS in a Protollin preparation may bedetermined by methods described herein and known in the art fordetermining the amount of LPS or OMPs that is free (i.e., uncomplexed)versus bound (i.e., in a OMP:LPS complex) such as capillaryelectrophoresis. LPS content of Protollin may be determined by a KDOassay, NMR, polyacrylamide gel electrophoresis and silver staining ofthe gel, and other methods practiced by a person having skill in theart. The OMP content of Protollin may be determined by any number ofassays that measure protein content including but not limited to massspectrometry methods such as LC-MS, reverse phase high pressure liquidchromatography (RP-HPLC), sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) (including protein staining such as withCoomassie blue or immunoblotting), N-terminal sequencing, amino acidanalysis, Lowry or BCA protein assays, and MALDI-TOFMS. Residual LPS ina Proteosome preparation may also be determined by the LPS assays, suchas KDO. Nucleic acids remaining in the OMP, LPS, or Protollinpreparation by methods known in the art to detect nucleic acids, and thepresence of detergent may be determined by HPLC.

In certain embodiments, the antigen is bacterial, for example, ananthrax Protective Antigen (PA) (see, e.g., Lindler et al., Infect.Immun. 66:5731-42 (1998)) or a plague antigen. The plague antigen usedin the immunogenic composition may comprise an F1 antigen or a V antigenfrom Yersinia pestis, or an F1-V antigen fusion protein antigen, or acombination thereof (see, e.g., Anderson et al., Infect. Immun.64:4580-85 (1996)). In other embodiments, the antigen is viral, such asa viral split antigen preparation (for example, a influenza splitantigen (see U.S. Patent Application 2004/0156867) or measles splitantigen). A viral split antigen is an antigen preparation that isseparated or isolated from a virus particle. A viral split antigengenerally comprises more than one single viral antigen and may compriseall viral antigens although not in the same proportion or quantity asmay be found in an intact virus particle. A split viral antigen may beprepared according to procedures that enrich one or more viral antigens,that is, the proportion of a particular antigen in a split antigenpreparation may be greater than in intact virus. For example, ainfluenza split antigen may be enriched for influenza Hemagglutininantigen.

Other exemplary microbial antigens include, but are not limited to,lipopolysaccharide, structural polypeptides or glycoproteins, flagellaror cilia proteins, toxins, virulence factors, viral core proteins, andviral envelope proteins and glycoproteins. In certain embodiments,isolated LPS may be an antigen, for example, LPS isolated from P.gingivalis, which may be formulated with Proteosomes for use instimulating an immune response to P. gingivalis for treating orpreventing gum disease, periodontal disease, tooth decay, and the like.

In certain embodiments, the immunogenic composition is administeredabout between one to about ten days, one to fourteen days, or one totwenty-one days after the immunostimulatory composition, preferably atleast three days after administration of the immunostimulatorycomposition, and elicits an adaptive immune response. Such a method fortreating or preventing a microbial infection may comprise administeringto a patient in need thereof an immunostimulatory composition havingProteosomes and liposaccharide in an amount and under conditionssufficient to elicit an innate or nonspecific protective immuneresponse; and administering to a patient in need thereof a immunogeniccomposition having Proteosomes, liposaccharide, and an antigen, or atleast two microbial antigens, in an amount and under conditionssufficient to elicit an adaptive immune response.

As described herein an innate immune response comprises host recognitionof invariant molecular constituents of infectious microorganisms thatrepresent molecular structures (PAMPs) shared by large groups ofmicroorganisms, for example, lipopolysaccharides with the conservedlipid A structure that are found in Gram-negative bacteria orpeptidoglycan common to Gram-positive bacteria. Such antigens arerecognized as non-self antigens by host receptors, thus the host elicitsa nonspecific immune response to destroy the non-self target. Thecapability of immunostimulatory compositions described herein, such asProtollin alone to stimulate innate immunity against aerosol challengewith various pathogens, such as Chlamydia trachomatis or Bacillusanthracis, may be determined according to methods described herein andknown in the art, including animal models. Animals, such as rodents(mice, rat, rabbits) can be treated with Protollin prepared as describedherein and then challenged with a pathogenic microorganism. Morbidityand mortality can then be determined. Animals may receive one, two,three, or more treatments with an immunostimulatory composition todetermine whether and for how long the innate immune response can bemaintained or re-stimulated. The capability of an immunostimulatorycomposition in the presence and absence of an immunogenic composition toelicit, enhance, or stimulate the innate immune response may also beexamined by the ability of the compositions to upregulate MHC class Iand II and B7.2 on peripheral blood B lymphocytes, dendritic, cells, andmucosal epithelial cells from wildtype mice and from TLR-2, TLR-4, andMyD88 knockout transgenic animals.

In one embodiment, the disclosure relates to a method for altering aninflammatory immune response, comprising administering to a subject (orpatient) in need thereof a therapeutically effective amount of animmunomodulatory composition, wherein the immunomodulatory compositioncomprises Proteosomes and liposaccharide, such that the inflammatoryimmune response is altered. In another embodiment, a method for treatingor preventing an allergic reaction comprises administering to a subject(or patient) in need thereof an amount of an immunomodulatorycomposition, wherein the immunomodulatory composition comprisesProteosomes and liposaccharide, such that the allergic reaction istreated, attenuated, ameliorated, or prevented. In certain embodimentswhen treating or preventing an allergic reaction, such as anallergen-induced reaction, the immunomodulatory composition comprisingProtollin will not contain a specific allergen, or if the allergen is abacteria, will not contain liposaccharide from the genus of bacteriathat is the allergen. That is, the Protollin need not have componentsthat are causing directly or indirectly an allergic reaction. In someembodiments, the Proteosomes and liposaccharide are obtained from thesame or different bacterial genera or species. Preferably, theProteosomes are from Neisseria species and the liposaccharide isobtained from Shigella, Chlamydia, Plesiomonas, Porphyromonas, or E.coli.

In another embodiment, after the immunomodulatory composition has beenadministered, a subject suffering from or at risk for an allergicreaction is given an amount of an immunogenic composition comprisingProteosomes, liposaccharide, and an allergen (e.g., microbial antigen orpollen) such that the allergic reaction is treated, prevented,diminished, attenuated, or ameliorated. In certain embodiments, theratio (wt:wt) (initial and/or post-detergent removal) of Proteosomes (orProtollin, which would include the combined weight of the Proteosomesand liposaccharide) to allergen (or antigen) of the immunogeniccomposition ranges from about 4:1 to about 1:4, preferably the ratio atleast 4:1 or at least 2:1. In other embodiments, the ratio ofProteosomes (or Protollin) to antigen of the immunogenic compositionranges from about 1:1 to about 1:500, preferably the ratio is at least1:20, at least 1:50, or at least 1:100. In certain other embodiments,the Proteosome or Protollin to allergen (or antigen) ratio in themixture ranges from about 1:1 to about 1:500, or in a range of about 1:1to about 1:200 or about 1:2 to about 1:200, or in a range of about 1:2to about 1:100, or in a range of about 1:5 to about 1:50, or in a rangeof about 1:2 to about 1:20. In certain embodiments, the allergen is atleast one of an inhaled particle, pollen (e.g., microspores of weeds,trees, grasses, etc.), vapor, gas, food, beverage (or a componentthereof), drug, toxin, microbial antigen (e.g., viral, viral splitantigen, bacterial, parasitic, fungal, and combinations thereof),dander, animal-derived compounds, dust (e.g., dust having LPS or dustmite feces), polypeptide, carbohydrate, nucleic acid, or any other agentcapable of eliciting an allergic reaction. The immunogenic compositionmay be administered about one to about ten days, one to twenty days, orone to thirty days after the immunomodulatory composition, or aboutthree days after, such that an inflammatory immune response or anallergic reaction is altered.

The immunostimulatory, immunogenic, and/or immunomodulatory compositionsdescribed herein may induce specific anti-antigen immune responses orimmunomodulatory effects, including one or more of the following. Aspecific humoral response may be elicited or stimulated that results inproduction of antigen specific antibodies, which may include any classof immunoglobulin, including IgG, IgA, IgM, and/or IgE, and isotypes ofthe classes. For example, the presence of specific IgG, IgA(particularly in mucosal secretions), and IgE in serum, nasal wash, lunglavage, or other tissues may be determined by any of a variety ofimmunoassays described herein and known in the art, including but notlimited to, ELISA, immunoblot, radioimmunoassay, immunohistochemistry,fluorescence activated cell sorting (FACS), Ochterlony, and the like.For detection of antigen or microorganism specific antibodies in animmunoassay, the biological sample may be permitted to interact with orcontact an antigen that is purified, isolated, partially isolated, or afragment thereof, or to interact with or contact a microorganism, whichmay be fixed (such as with ethanol or formaldehyde) or unfixed ornon-denatured. Mucosal secretions include those collected from therespiratory tract, including the nasopharynx and lungs. Functionalassays may also be performed, such as the ability of an antigen-specificantibody to neutralize a toxin (such as a macrophage protection assay),facilitate phagocytosis or opsonization of a microorganism, or toprevent entry of a microorganism into a host cell, or to prevent entry,fusion, or propagation of a microorganism such as a virus in a hostcell. Such methods are described herein and are routinely practiced byskilled artisans.

Cell-mediated immunity (CMI) or immune response in a subject who hasreceived one or more of the immune compositions described herein mayalso be determined using methods described herein and known in the art.A cell mediated immune response includes determining whether an immuneresponse has shifted from a predominantly Th2 response to a balanced ormixed Th1 and Th2 response (due to a an increase in Th1 response orconcomitant increase in Th1 and decrease in Th2 response), or to apredominantly Th1 response. Similarly, a shift from a Th1 response to abalanced or mixed Th1/Th2 response or an increased or predominant Th2response may be determined. For example, levels of Th1 cytokines, suchas IFN-γ, IL-2, and TNF-β, and Type 2 cytokines, such as IL-4, IL-5,IL-9, IL-10, and IL-13, may be determined according to methods describedherein and practiced in the art, including ELISA, ELISPOT, and flowcytometry (to measure intracellular cytokines). Type 1 responses arepredictive of induction of other CMI-associated responses, such asdevelopment of cytotoxic T cells (CTLs), which are indicative of Th1immunity. Immune cell proliferation and clonal expansion resulting froman antigen-specific elicitation or stimulation of an immune response maybe determined by isolating lymphocytes, such as spleen cells or cellsfrom lymph nodes, stimulating the cells with antigen, and measuringcytokine production, cell proliferation and/or cell viability, such asby incorporation of tritiated thymidine or non-radioactive assays, suchas MTT assays and the like.

In any of these aforementioned methods, the immunomodulatorycompositions, immunostimulatory compositions, and the immunogeniccompositions may further comprise a pharmaceutically acceptable carrier,excipient, or diluent as described herein. A pharmaceutical compositionmay be a sterile aqueous or non-aqueous solution, suspension oremulsion, which additionally comprises a physiologically acceptablecarrier (i.e., a non-toxic material that does not interfere with theactivity of the active ingredient). Such compositions may be in the formof a solid, liquid or gas (aerosol). Alternatively, compositions of thepresent invention may be formulated as a lyophilizate. Pharmaceuticalcompositions within the scope of the present invention may also containother components, which may be biologically active or inactive. Suchcomponents include, but are not limited to, buffers (e.g., neutralbuffered saline or phosphate buffered saline), diluents, stabilizers,dyes, flavoring agents, and suspending agents and/or preservatives.

Any suitable carrier known to those of ordinary skill in the art may beemployed in the pharmaceutical compositions of the present invention.Carriers for therapeutic use are well known, and are described, forexample, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro ed. (1985)). In general, the type of carrier is selectedbased on the mode of administration. Pharmaceutical compositions may beformulated for any appropriate manner of administration, including, forexample, topical, oral, nasal, intrathecal, rectal, vaginal, sublingualor parenteral administration, including subcutaneous, intravenous,intramuscular, intrastemal, intracavernous, intrameatal or intraurethralinjection or infusion. For parenteral administration, the carrierpreferably comprises water, saline, alcohol, a fat, a wax or a buffer.For oral administration, any of the above carriers or a solid carrier,such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose, ethyl cellulose, glucose, sucroseand/or magnesium carbonate, may be employed.

A pharmaceutical composition (e.g., for oral administration or deliveryby injection) may be in the form of a liquid (e.g., an elixir, syrup,solution, emulsion or suspension). A liquid pharmaceutical compositionmay include, for example, one or more of the following: sterile diluentssuch as water for injection, saline solution, preferably physiologicalsaline, Ringer's solution, isotonic sodium chloride, fixed oils such assynthetic mono or diglycerides which may serve as the solvent orsuspending medium, polyethylene glycols, glycerin, propylene glycol orother solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. A parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. The use of physiological saline is preferred,and an injectable pharmaceutical composition is preferably sterile.

As used herein, the terms treat and ameliorate refer to the therapeuticadministration of a desired composition or compound, in an amount andunder conditions sufficient to treat, inhibit, attenuate, ameliorate,reduce, prevent or alter at least one aspect or marker of a disease, ina statistically significant manner or in a clinically significantmanner. A therapeutically effective amount of an immunomodulatorycomposition, immunostimulatory composition, or immunogenic compositionis the amount of the composition that treats at least one aspect ormarker of a disease as described herein.

The compositions described herein that comprise one or moreimmunomodulatory composition, immunostimulatory composition, andimmunogenic composition may be in any form that allows the compositionto be administered to a subject, such as a human or animal. For example,compositions may be prepared and administered as a liquid solution orprepared as a solid form (e.g., lyophilized), which may be administeredin solid form, or resuspended in a solution in conjunction withadministration. The compositions may be formulated to allow the activeingredients contained therein to be bioavailable upon administration toa subject or patient or may be bioavailable via slow release.Compositions that will be administered to a subject or patient take theform of one or more dosage units, for example, a drop may be a singledosage unit, and a container of one or more compositions may hold aplurality of dosage units. In certain preferred embodiments, any of theaforementioned pharmaceutical compositions comprising animmunostimulatory composition, or an immunostimulatory composition withan immunogenic composition that comprises at least one antigen (orimmunogen) or a cocktail of immunogens, or an immunomodulatorycomposition are in a container, preferably in a sterile container.

The design of a particular protocol for administration, including dosagelevels and timing of dosing are determined by optimizing such proceduresusing routine methods well known to those having ordinary skill in theart. Pharmaceutical compositions may be administered in a mannerappropriate to the disease to be treated (or prevented). An appropriatedose and a suitable duration and frequency of administration will bedetermined by such factors as the condition of the patient, the type andseverity of the patient's disease, the particular form of the activeingredient and the method of administration. In general, an appropriatedose and treatment regimen provides the compositions in an amountsufficient (therapeutically effective amount) to provide therapeuticand/or prophylactic benefit (e.g., an improved clinical outcome, such asmore frequent complete or partial eradication of the infection, orlonger disease-free and/or overall survival, or a lessening of symptomseverity). For prophylactic use, a dose should be sufficient to prevent,delay the onset of, or diminish the severity of a disease associatedwith the particular infectious microorganism.

In one embodiment, any one of the immunomodulatory composition,immunostimulatory composition or immunogenic composition is administerednasally. Other routes of administration include enteral, parenteral,transdermal/transmucosal, sublingual, nasal, and by inhalation. The termenteral, as used herein, is a route of administration in which theimmunogenic composition is absorbed through the gastrointestinal tractor oral mucosa, including oral, rectal, and sublingual. The termparenteral, as used herein, describes administration routes that bypassthe gastrointestinal tract, including intraarterial, intradermal,intramuscular, intranasal, intraocular, intraperitoneal, intravenous,subcutaneous, submucosal, and intravaginal injection or infusiontechniques. The term transdermal/transmucosal, as used herein, is aroute of administration in which any of the compositions describedherein is administered through or by way of the skin, including topical.The terms “nasal” and “inhalation” encompass techniques ofadministration in which an immunogenic composition is introduced intothe pulmonary tree, including intrapulmonary or transpulmonary.Preferably, the compositions of the present invention are administerednasally.

Furthermore, the immunogenic compositions of this invention can be usedto enhance immunity, or as a follow on immunization or toleranceinduction, when given together with another vaccine, such as a liveattenuated vaccine, or a non-live, subunit vaccine. For example,compositions comprising one or more antigen or fragment or fusionthereof with Projuvant or Protollin may be used as a priming or boostingimmunization (by mucosal or parenteral routes) prior to or subsequent toadministering a different vaccine.

All U.S. patents, U.S. patent application publications, U.S. patentapplications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference, intheir entirety. The invention having been described, the followingexamples are intended to illustrate, and not limit, the invention.

EXAMPLES Example 1 Preparartion of Proteosomes

Immunogens (e.g., microbial antigens or allergens) may be formulatedwith Proteosomes to form an immunogenic composition of the instantinvention capable of eliciting a protective immune response or tolerancein a human or animal subject. Proteosomes are useful as an adjuvant andare comprised of outer membrane proteins purified from Gram-negativebacteria. Methods for preparing Proteosomes are described in, forexample, Mallett et al. Infect. Immun. 63:2382, 1995; U.S. Pat. No.6,476,201 B1; U.S. Patent Application Publication No. 2001/0053368; andU.S. Patent Application Publication No. 2003/0044425. Briefly, a pasteof phenol-killed Group B type 2 Neisseria meningitidis was extractedwith a solution of 6% Empigen® BB (EBB) (Albright and Wilson, Whithaven,Cumbria, UK) in 1 M calcium chloride. The extract was precipitated withethanol, solubilized in 1% EBB-Tris/EDTA-saline, and then precipitatedwith ammonium sulfate. The precipitated Proteosomes were re-solubilizedin 1% EBB buffer, diafiltered, and stored in a 0.1% EBB buffer at −70°C.

A flow chart of this process, which resulted in Proteosomes having aliposaccharide content of between about 0.5% and about 5%, is shown inFlowchart 1A (FIG. 1A). Proteosomes may also be prepared by omitting theammonium sulfate precipitation step to shorten the process. Theresultant Proteosomes having a liposaccharide content of between about12% and about 25%, and may, depending upon the materials, be betweenabout 15% and about 20%, as shown in Flowchart 1B (FIG. 1B). A personhaving ordinary skill in the art could adjust methods for preparingformulations comprising Projuvant or OMP-LPS (Protollin) compositions asdescribed herein to optimize particular characteristics of the vaccinecomponents.

Example 2 Preparation of Liposaccharides

The example in Flowchart 2 (FIG. 2) shows the process for the isolationand purification of LPS (e.g., non-detoxified) from S. flexneri or P.shigelloides. This process can similarly be used for preparing LPS fromone or more other Gram-negative bacteria, including Shigella,Plesiomonas, Porphyromonas, Escherichia, and Salmonella species.Following growth of bacteria by fermentation in 300 L, the bacteria weresedimented and the cell paste was re-hydrated with 3 ml 0.9 M NaCl,0.005 M EDTA, and 10 mg lysozyme per gram of bacterial paste. Lysozymedigestion was allowed to proceed for 1 hour at room temperature. Then 50U/ml Benzonase® (DNase) (Merck Chemicals) in 0.025 M MgCl₂ was added,and DNase digestion was allowed to proceed at room temperature for 30minutes. The suspension was then cracked by passage through amicrofluidizer at 14,000 to 19,000 psi. Fresh DNase (50 U/ml) was added,and digestion of the suspension was allowed to proceed for an additional30 minutes at room temperature. The digested cell suspension was heatedto 68° C. in a water bath. An equal volume of 90% phenol (also heated to68° C.) was then added, and the mixture was incubated with shaking at68° C. for 30 minutes. The mixture was centrifuged at 4° C. to separatethe aqueous and organic phases. The aqueous phase was harvested and theorganic phase was re-extracted with WFI (water for injection) at 68° C.for 30 minutes. The mixture was centrifuged at 4° C., the second aqueousphase was harvested, and the two harvested aqueous phases were combined.To precipitate nucleic acids, 20% ethanol with 10 mM CaCl₂ was added tothe pooled aqueous phases. The mixture was stirred at 4° C. overnight.Precipitated nucleic acids were then sedimented by centrifugation at10,000×g for 30 minutes. The supernatant was harvested, concentrated,and diafiltered using a 30,000 MW hollow fiber cartridge into 0.15 MNaCl, 0.05 M Tris, 0.01 M EDTA, and 0.1% Empigen® BB, pH 8.0 (TEENbuffer). The LPS was then sterile-filtered using a 0.22 μm Millipak® 60filter unit, aliquoted into sterile storage containers, and frozen at−80° C. Stability studies indicated that bulk LPS has a storage life ofat least 2 years.

Example 3 Preparation and Characterization of Proteosome:LiposaccharideAdjuvant

A Proteosome adjuvant formulation was prepared by admixing Proteosomeswith LPS (Protollin). The LPS can be derived from any of a number of oneor more Gram negative bacteria, such as Shigella, Plesiomonas,Escherichia, or Salmonella species (see Example 2), which is mixed withthe Proteosomes of Example 1, as described in Flowchart 3 (FIG. 3).Briefly, Proteosomes and LPS were thawed overnight at 4° C. and thedetergent concentration was adjusted to 1% Empigen® BB in TEEN buffer.The Proteosomes and LPS were mixed for 15 minutes at room temperature inquantities that resulted in a final wt/wt ratio of between about 10:1and about 1:3 of Proteosome:LPS. The Proteosome:LPS mixture wasdiafiltered on an appropriately sized (e.g., Size 9) 10,000 MWCO(molecular weight cut-off) hollow fiber cartridge into TNS buffer (0.05M Tris, 150 mM NaCl pH 8.0). The diafiltration was stopped when Empigen®content in the permeate was <50 ppm, which was determined by Empigen®Turbidity Assay or by a Bradford Reagent Assay manufacturer's andstandard protocols. The bulk adjuvant (referred to herein as OMP-LPS)was concentrated and adjusted to 5 mg/ml protein. The protein contentwas determined by a standard Lowry assay. The adjuvant wassterile-filtered using a 0.22 μm Millipak 20 filter unit. The bulkadjuvant was aliquoted into sterile storage containers and frozen.

The OMP-LPS adjuvant was tested for (1) Empigen® (400 ppm) usingreverse-phase HPLC; (2) protein content by a Lowry assay; and (3) LPScontent by measurement in a 2-keto-3-deoxyoctonate (KDO) assay. TheOMP-LPS composition was further characterized for particle sizedistribution as determined by quantitative number weighted analysisusing a particle seizer (e.g., Brookhaven Instruments model 90 plus orsimilar machine) (10-100 nm). However, the particle size for the complexmay increase or modulate with varying (e.g., higher) Proteosome to LPSratio. These Proteosome:LPS complexes have been termed Protollin.Stability data indicated that this formulation is stable for longer than2 years.

Protollin has been prepared using other sources of LPS. Two Protollinpreparations were made using LPS from two different strains of E. coliand had similar adjuvant activity. Protollin is also prepared using N.meningiditis LPS. N. meningitis LPS is frequently called LOS denotinglipooligosaccharide because the O-side chain of N. meningiditisliposaccharide is shorter than that of other Gram-negative bacteria suchas E. coli and Shigella. Production of Protollin with N. meningiditisLPS (Protollin-Nm) is different from all other versions of Protollin.During the production of N. meningiditis Proteosome OMPs, LPS is removedby ammonium sulfate precipitation techniques so that Proteosomeparticles have less than 2.5% N. meningiditis LPS. If the LPS is notremoved at this step, the resultant Proteosome particles have about20-25% LPS, resulting in an OMP:LPS ratio ranging from about 5:1 toabout 4:1. Thus, Protollin-Nm is produced in a single step, therebyeliminating further purification of the Proteosome particles. An aliquotof each Protollin is retained for use in, for example, a spin-down assayto verify Proteosome OMP complexing with LPS. Each of these versions ofProtollin is tested in mice for adjuvant activity after formulation withrPA (recombinant Protective Antigen).

Example 4 Immunization with Protollin Formulated with Plague AntigenF1-V

This Example describes the ability of Proteosome:LPS (Protollin)compositions formulated with plague antigen (F1-V) to elicit an immuneresponse capable of protecting against a lethal challenge with Yersiniapestis. The F1-V immune response was assessed by immunizing groups of 206-8 week old female Swiss-Webster mice (Charles River, St-Constant,Quebec) on days 0 and 21. Freshly thawed aliquots of Protollin and F1-Vfusion protein (U.S. Army Medical Research Institute of InfectiousDisease) solutions were mixed less than 16 hours prior to immunization.For nasal administration, mice were first lightly anesthetized byisoflurane inhalation. Twenty-five microliters of vaccine or appropriatecontrol samples (Protollin alone or F1-V alone) were applied to thenares (12.5 μl per nostril) of each mouse. In parallel, a group of micewere immunized intramuscularly (i.m.) by injection into hind limbs with25 μl F1-V adsorbed to 500 mg of Alhydrogel®. Control i.m. injectionswere also performed. Thirty-five and 55 days thereafter, 10 mice fromeach group were euthanized by asphyxiation with CO₂ and exsanguination.Serum, nasal wash, and lung lavage samples were obtained and stored at−80° C. Spleens were processed for in vitro restimulation and assessmentof released cytokines. The remaining 10 mice from each group werechallenged on day 35 or 55 by inhalation of 170-250 LD₅₀ of aerosolizedY. pestis (Colorado 92 strain) to assess protection. Mice were monitoredfor 28 days after challenge for determination of morbidity andmortality.

Antibodies present in serum and lung lavage fluid samples were obtainedfrom mice immunized intranasally with two doses of F1-V antigenformulated with Protollin and compared with samples from mice immunizedintranasally with F1-V alone or with mice immunized intramuscularly withAlhydrogel®-adsorbed F1-V. The results are shown in FIG. 4. Allcombinations of Protollin and F1-V were highly immunogenic and elicitedF1-V specific serum IgG titers of between 1 and 9 mg/ml (FIG. 4A). Onboth sampling days a trend towards lower titers elicited by the lowerF1-V and/or Protollin concentrations was observed, but no significantdifferences were measured in the specific IgG titers elicited by anycombination of F1-V and Protollin concentrations or those elicited byintramuscular injection of 20 μg of F1-V adsorbed onto Alhydrogel(P>0.05). All specific serum IgG titers in mice immunized with F1-Vformulated vaccines were significantly higher than titers measured inanimals that received nasal administration ofunformulated F1-V controls(P≦0.001). No F1-V specific antibodies were detected in serum fromcontrol mice.

The levels of specific anti-F1-V, anti-F1, and anti-V antibodies presentin lung lavage samples were determined by ELISA performed according tostandard methodologies using F1-V fusion protein, F1 polypeptide, and Vpolypeptide as antigens (U.S. Army Medical Research Institute ofInfectious Disease). IgG and IgA antibody titers were determined onindividual samples by ELISA as previously described (Plante et al.,Vaccine 20:218 (2001)). Briefly, ELISA plates were coated with F1-V, F1,or V at pre-determined concentrations. Bound antibody is detected withHRP-conjugated anti-mouse IgG or IgA. Data are expressed as geometricmeans of antibody concentrations in individual mouse samples, and thesignificance of the data is assessed by ANOVA analysis usingTukey-Kramer pair-wise comparisons. All groups of mice immunizedintranasally with F1-V antigen plus Protollin had high titers of F1-Vspecific lung IgA as shown in FIG. 4B, confirming that immunization bymucosal (e.g., intranasal) routes efficiently elicits mucosalantibodies. ANOVA analysis indicated no significant differences in theIgA titers among groups of mice that were immunized with differentcombination of F1-V plus Protollin. Animals that received unformulatedF1-V alone nasally had barely detectable IgA levels. Secretory IgA wasnot detected in samples from mice injected i.m. with Alhydrogel-adsorbedF1-V.

Sera and lung lavage fluid from all mice immunized with a 20 μg dose ofF1-V antigen were examined in an ELISA to determine if antibodies insera specifically bound to F1 or V or both components. In all instancesand at both sampling times, serum IgG and lung lavage fluid IgAantibodies that recognized F1 and V portions of the F1-V antigen weredetected (Table 1). Binding of lung lavage and serum antibodies frommice immunized with Protollin compositions to the F 1 and V portions ofthe F1-V antigen indicated that the immune response was primarilydirected against the V component of the F1-V fusion protein (Table 1).Lung lavage samples also contained significant titers of F1-V specificIgG, even though the titers represented only a small percent of theserum titers (range 0.11%-0.56%; median 0.175%),

TABLE 1 Ratio of Anti-F1 to anti-V Antibodies in Serum and Lung LavageFluids of Mice Immunized with Several Formulations of Plague AntigenF1-V F1-V + F1-V + F1-V + F1-V + 2.5 μg 1 μg 0.25 μg F1-V AlhydrogelProtollin Protollin Protollin i.n. i.m. Serum IgG d35 0.31 0.30 0.340.31 0.65 Serum IgG d55 0.25 0.26 0.33 0.58 0.29 Lung IgA d35 0.42 0.390.33 N/A N/A Lung IgA d55 0.49 0.29 0.32 N/A N/A Lung IgG d35 0.40 0.530.44 N/A 1.02 Lung IgG d55 0.48 0.48 0.46 N/A 1.03

Example 5 Determination of Cytokine Profile After Immunization withProtollin:Plague Antigen

To compare the phenotype (type 1 or type 2) of the adaptive immuneresponse elicited by intranasally administered Protollin or injectedAlhydrogel® adjuvanted F1-V vaccine, splenocytes from selected groups ofimmunized mice (see Example 4) were re-stimulated in vitro with F1-V.Spleens from each group of mice were pooled and processed into singlecell suspensions according to standard methods. The splenic cellsuspensions were then incubated with different concentrations of F1-V.Cytokines released into culture supernatants were determined byquantitative ELISA using OptEIA kits (BD Biosciences, San Jose, Calif.).The amounts of IFN-γ, TNF-α, and IL-5 cytokines released into culturesupernatants were determined. Splenocytes from mice immunizedintranasally with F1-V (50 μg) mixed with Protollin (1 μg) responded toin vitro re-stimulation by secreting high levels of both IFN-γ andTNF-α; a very low amount of IL-5 was also detected. In contrast,splenocytes from mice immunized by injection of F1-V (20 μg) adsorbedonto Alhydrogel responded by secreting comparatively lower amounts ofIFN-γ and TNF-α, although a significant amount of IL-5 was detected.Thus, the cytokine profile elicited by nasal administered of Protollinformulated (adjuvanted) with F1-V antigen was consistent with elicitinga type 1 immune response, whereas the cytokine profile induced by i.m.injection of F1-V antigen formulated with Alhydrogel® is more consistentwith a response biased toward a type 2 phenotype.

Example 6 Challenge of Immunized Mice with Aerosolized Live Y. Pestis

This Example describes immune protection provided by intranasalimmunization with F1-V formulated with Protollin. Mice that receivedF1-V combined with Protollin were challenged by whole-body exposure tolive aerosolized Y. pestis (see Example 4). The level of protection fromchallenge indicated by survival of animals was compared with protectionof mice that were injected with F1-V adsorbed onto Alhydrogel and micethat received intranasal administration of F1-V alone or Protollinalone. On day 35 and at a challenge dose of 169 LD₅₀ Y. pestis, miceimmunized intranasally with 5, 20, or 50 μg of F1-V plus 1 or 2.5 μg ofProtollin all survived, as did mice injected with F1-V adsorbed ontoAlhydrogel. Survival of mice immunized nasally with 5, 20, or 50 g ofF1-V and 0.25 μg of Protollin was 90%, 100%, and 90%, respectively,while survival of mice immunized nasally with the same doses of F1-Vwithout Protollin was only 30%, 40% and 40%, respectively. None of thecontrol mice that received Protollin alone survived longer than 4 dayspost challenge. Survival for all mouse groups immunized with F1-Vformulated with Protollin was highly significant compared to survival incontrol mice or mice immunized with F1-V alone (P≦0.05 or better usingFisher's Exact Probability Test). The results for mice immunized with 20μg doses of F1-V are shown in FIG. 5A, and the results for animalsimmunized with 5 μg doses of F1-V are shown in FIG. 5D.

Similar results were obtained when animals were challenged on day 55(FIG. 5B). All mice immunized with 2.5 μg Protollin formulated with F1-Vand mice immunized by injection of F1-V adsorbed onto Alhydrogelsurvived challenge by Y. pestis. All mice immunized with 1 μg ofProtollin formulated with 50 μg or20 μg of F1-V also survived, while 90%of animals that received all other combinations of Protollin and F1-Vsurvived. In all mice immunized with formulated F1-V (F1-V plusProtollin), the observed protection was highly significant (P≦0.01 orbetter) compared to mice immunized with unformulated F1-V (10-30%protection) or the Protollin only control group of mice in which noanimals survived.

Mice immunized nasally with 50 μg of F1-V with or without 1 μg ofProtollin, or that were injected with 20 μg of F1-V adsorbed ontoAlhydrogel, were challenged on day 55 by whole body exposure to 254 LD₅₀aerosolized live Y. pestis. The results are presented in FIG. 5C. Eightypercent of the mice that were immunized with 50 μg F1-V plus 1 μgProtolin survived; 60% of mice that were immunized with 20 μg of F1-Vadsorbed onto Alhydrogel survived; and 20% of animals that that receivedF1-V only survived lethal challenge. Control mice given Protollin aloneall died. Immunization with formulated F1-V induced significantprotection against death compared to control mice (P≦0.001 for nasalF1-V plus Protollin; P≦0.01 for i.m. injected F1-V). Nasal immunizationwith F1-V plus Protollin offered significantly protection against deaththan immunization with F1-V alone (P≦0.05). Survival of mice injectedwith F1-V adsorbed onto Alhydrogel was not significantly better thansurvival of animals immunized intranasally with F1-V without Protollin(P=0.095).

Example 7 Protection of Mice by Protollin Anthrax ImmunogenicFormulations

In this Example, Protollin formulated with Protective Antigen (PA) ofBacillus anthracis (see Example 8) was assessed for its capability toinduce an immune response exemplified by a statistically significantreduction in PA-mediated macrophage killing. Mice were immunized nasallyon days 0 and 14 with 5 or 25 μg rPA (List Biological Laboratories)admixed with 1 μg of Protollin.

A standard ELISA protocol was used to detect IgG and IgA I serum andlung lavage samples. Briefly, serial dilutions of the test samples(serum and lavage fluids) were added to the wells of ELISA plates thatwere coated with purified rPA. Antigen-specific antibodies that adheredto the immobilized antigen were detected with anti-mouse constant regionantibody conjugated to horseradish peroxidase (HRP). Followingincubation of the HRP antibody conjugate, the wells were washed, TMBsubstrate was added, and the amount of bound HRP antibody was detectedby measuring absorbance at 490 nm. Antibody concentrations in the testsamples were calculated from standard curves that were run in parallel,using purified standard antibodies for IgA and IgG. ELISA data wereexpressed as geometric means at 95% confidence levels according to astatistical analysis using log-transformed data. Animals that receivedProtollin plus PA showed specific anti-PA serum IgG and lung IgA levelsthat were significantly higher than those of mice that were intranasallyadministered 5 or 25 μg of rPA alone (p<0.05) (FIG. 6A-B). Mucosal IgAlevels in animals treated with the Protollin alone or rPA alone werebelow the detection level of this assay.

The capability of specific anti-PA antibodies to neutralize PA-mediatedmacrophage killing was evaluated using a cell culture assay system usingserum and lung lavage fluid samples from the animals. RAW264.7macrophages (ATCC, Manassas, Va.) (2×10⁵ cells per well) were plated insterile 96-well plates and incubated at 37° C. for 24 hours in 5% CO₂.Serial dilutions of serum or lung lavage fluid samples from thePA-immunized animals were incubated with a PA solution (4 μg/ml in rPMIcell culture media supplemented with 10% fetal bovine serum) 1 hour at37° C., after which the mixtures were added to the wells containingRAW264.7 cells. A solution of Lethal Factor (LF) was added to the wells,and the plates were incubated at 37° C. in 5% CO₂ for 4 hours. Asolution of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) (Sigma-Aldrich, St. Louis, Mo.) to measure cell viability wasthen added to each well, and the plates incubated for 4 hours at 37° C.in 5% CO₂. The reaction was stopped by adding 20% SDS in 50% DMF(dimethylformamide), pH 4.3. Optical density is measured with an ELISAplate reader (Molecular Devices, Menlo Park, Calif.) at 570 nm(reference at 690 nm). The assay is linear in for cell concentrations inthe range of 10⁴ to 10⁵ cells/well. The nasal vaccine, rPA+Protollin,elicited comparable levels of antibodies that neutralized rPA activityas did the IM alum-adjuvanted vaccine (FIG. 7).

Example 8 Preparation of Anthrax Vaccine Formulations

Nasal Protollin anthrax vaccines are made by admixing the anthrax PAantigens with soluble pre-formed Proteosome plus LPS (i.e., Protollin)prior to immunization. Both rPA and rPA-anch (rPa with a hydrophobicanchor sequence) antigens are evaluated with several differentformulations of Protollin to determine the formulation(s) with preferredimmunogenic antigen and Protollin components. Control formulationsconsist of, for example, Protollin alone or mixed with at least one andpreferably two control antigens, including a recombinant streptococcalprotein with or without a hydrophobic anchor sequence (anch).Accordingly, formulations of Protollin that are evaluated have differentsources of LPS, varied Proteosome:LPS ratios, and varied Protollin:rPAantigen ratios. rPA-anch is also formulated with Proteosome proteinsthat have very low levels of LPS (<2% by weight) using the dialysis ordiafiltration methodology described herein, which is designed to removeor reduce the concentration of detergent in which the Proteosomeadjuvant is stored. These Proteosome adjuvant preparations do not haveexogenous LPS added. The Proteosome preparations used to formulateProtollin as described in this example have been used in extensivepre-clinical toxicity studies as well as Phase 1 and Phase 2 humanclinical trials to evaluate safety, immunogenicity, and efficacy of aProteosome nasal influenza vaccine.

The preferred LPS bacterial type and source and the preferred ratio ofOMP:LPS formulation is determined by immunogenicity studies. Afterfermentation of the preferred bacteria, LPS is purified and analyzed.The purified LPS is then mixed with Proteosome OMP particles at theselected ratio to form a OMP:LPS complex, Protollin. The extent ofcomplex formation of the LPS and OMPs is determined according to“free-vs.-bound” assays using capillary electrophoresis, LPS “spiking”studies, and other analyses practiced in the art. Protollin is analyzedfor LPS content using KDO, NMR, and silver stain PAGE, and analyzed forProteosome OMP content using LC-MS, RP-HPLC, SDS-PAGE (Coomassie Bluestain & Western immunoblot using monoclonal and polyclonal antibodies),N-terminal sequencing, amino acid analysis, total protein by Lowry orBCA, and MALDI-TOFMS for example. The presence of residual LPS, nucleicacids, and detergents is determined using various techniques includingKDO to determine LPS content and HPLC to determine the presence ofdetergent.

Example 9 Evaluation of Serum and Mucosal Immune Response

An ELISA is performed to determine total immunoglobulin and B. anthracisProtective Antigen (PA)-specific IgG, IgA, and IgM titers in biologicalsamples obtained from animals (mice, rabbits) immunized with testimmunostimulatory or immunogenic formulations as described herein.Samples include serum, nasal and lung mucosal washes. A standard ELISAprotocol is used to determine linearity, specificity, sensitivity, andreproducibility. Briefly, serial dilutions of the test samples (serumand lavage fluids) are added to the wells of ELISA plates that arecoated with purified rPA, or derivatives thereof. Antigen-specificantibodies that adhere to the immobilized antigen are detected withanimal (for example, and anti-rabbit or anti-mouse constant regionantibody) and antibody-subtype specific horseradish peroxidase (HRP)conjugated antibodies. Following incubation of an HRP antibodyconjugate, the wells are washed, TMB substrate is added, and the amountof bound HRP antibody is detected by measuring absorbance at 490 nm.Antibody concentrations in the test samples are calculated from standardcurves that are run in parallel, using purified standard antibodies forIgA, IgM, and/or IgG (including mouse isotypes, IgGI and IgG2a). Whenappropriate, specific antibody levels in mucosal wash fluid samples arestandardized and normalized by expressing the specific antibodiesdetected in comparison to the total amount of IgA or IgG in the sample.ELISA data are expressed as geometric means at 95% confidence levelsaccording to a statistical analysis using log-transformed data.

Example 10 Macrophage Protection Assay to Evaluate for AnthraxNeutralizing Antibodies

This Example describe an assay that is used to measure neutralizingantibodies from animals immunized with Proteosome vaccines. In vitroassays are designed to measure inhibition of anthrax toxin cytotoxicityby serum samples obtained from immunized animals. Serial dilutions ofthe serum samples are added in combination with lethal quantities ofanthrax PA and Lethal Factor (LF) (List Biological Laboratories,Cambell, Calif.) to J774A.1 macrophage cells or other macrophage cellline for 3 hours at 37° C. in a 96-well plate. Cell viability ismeasured chromatographically by adding one-tenth volume solution of 5mg/ml MTT. After an incubation of 4 h at 37° C., the assay plates areanalyzed spectrophotometrically at 570 nm using an ELISA plate reader(Molecular Devices, Menlo Park, Calif.). The assay is linear for cellconcentrations in the range of 10⁴ to 10⁵ cells/well.

Example 11 Cell Mediated Immunity Assay to Evaluate Immunization AgainstAnthrax

Cellular immune responses that are induced following immunization withthe Proteosome-based PA vaccines are studied using a variety of methods.For example, T cell-derived cytokines are assessed on PA-re-stimulatedpurified or enriched T cells isolated from mouse spleen and/ormediastinal lymph nodes. The presence and levels of Type 1 (e.g., IFN-γ)and type II (e.g., IL-4 and IL-5) cytokines are determined by one ormore methods including ELISA, ELISPOT. The presence and levels ofintracellular cytokines is determined by standard flow-cytometrymethods. Techniques that measure proliferation of PA-re-stimulated PBMCT cells are used to evaluate cell-mediated immune responses in rabbitsdue to unavailability of reagents that are specific for rabbitcytokines.

T lymphocyte proliferation assays are used to measure the effect ofimmunization on clonal expansion and determine the presence of memorylymphocytes in animals from various animal models. Following animalsacrifice, mediastinal and cervical lymph nodes are surgically removedusing standard techniques, the lymphocytes are isolated and thencultured with and without PA. Proliferation is measured by uptake of³H-thymidine. Cells from animals immunized with sham vaccine will beused as negative controls. Assay results are used to determine theeffect of immunization on T cell differentiation in the lymph nodes,particularly in relation to mucosal immunity. The results are alsocorrelated with efficacy of immunization determined in anthrax challengeanimal studies.

Example 12 Methods for Collecting Nasal Wash and Lung Lavage

Lung and nasal washes from mice and rabbits were collected to analyzethe immune response to immunostimulatory or immunogenic formulations. Inmice, nasal washes and lung lavage were performed by cannulating thetrachea and pumping 1 ml PBS supplemented with 0.1% bovine serum albuminand protease inhibitors (General Use Protease Inhibitor Cocktail;Sigma-Aldrich Chemicals containing 0.2 mM AEBSR, 1 μg/ml aprotinin, 3.25μM bestatin, 10 μM Leupeptin) upwards through the trachea. Fluidemerging from the nostrils was collected, vortexed, and then centrifugedto remove tissue and cell debris. The supernatants were stored at −70°C. A cannula was reinserted into the trachea and directed toward thelung for collection of lung fluids. Lungs were 1avaged twice with 1.0 mlprotease supplemented PBS; the fluid was collected and vortexed; and thecell debris removed by centrifugation. Lung and nasal washes were storedat −70° C. Rabbit mucosal fluids were similarly collected, adjusting thevolumes as appropriate. In certain experiments, after collecting mucosalsamples, cervical and mediastinal lymph nodes were surgically removedand mononuclear cells isolated and cultured for ELISPOT antibody,cytokine, and CMI assays as described herein.

Example 13 Eliciting Innate Immunity in Rabbits with Protollin

Protollin has adjuvant activity related to both Proteosomes and LPS.Capability of Protollin alone (without antigen) to stimulate innateimmunity against aerosol challenge with various pathogens, such asChlamydia trachomatis or Bacillus anthracis, is determined. Rabbits arechallenged via aerosol with 100 or 200 LD₅₀ of anthrax spores within afew days, or longer, after receiving Protollin. Rabbits that survive theanthrax challenge are immunized again with Protollin to determine ifprotection can be prolonged or innate immunity restimulated.

Example 14 Induction of an Immune Response by Proteosome:LPSFormulations Against Challenge with Chlamydia Trachomatis

Chlamydia LPS is highly conserved among various Chlamydia strains anddata suggest that antibodies specific for Chlamydia-genus specific LPSmay protect against Chlamydia infection (Peterson et al., Infect. Immun.66: 3848, 1998). Chlamydia LPS was produced in a recombinant Escherichiacoli that synthesizes both E. coli LPS and a rough ‘chlamydia-like’mutant (rLPS) in equal proportions (C.t./E. coli rLPS) (purchased fromGlycoTech, Kukels, Germany). A murine lung model for Chlamydia infectionwas used to evaluate the immune response effected by C.t./E. coli rLPSformulated with Protollin.

Proteosome:LPS compositions were prepared using a dialysis procedurewith C.t./E. coli rLPS. Proteosomes alone (solubilized in 0.1% Empigen®BB detergent) or solubilized with LPS (solubilized in 1% Empigen BB®detergent) at a final concentration of 1 mg/ml Proteosomes were added toSpectraPor® dialysis tubing with a 1,000 MW cutoff. Dialysis wasperformed against phosphate buffered saline (PBS) for 10 days or longer.The duration of dialysis can be adjusted to retain varying amounts ofdetergent in the vaccine formulation including, for example,concentrations from 250, 500, 750, 1000 ppm, or more, or even loweramounts (e.g., 50 ppm). The concentration of LPS in the dialyzed sampleswas determined by measuring 3-Deoxy-D-manno-octulosonate, and theconcentration of protein was determined using the standard Lowry method.A mixture of Proteosomes and C.tr./E. coli LPS prior to dialysis wasapproximately at a ratio of 1:2.7 (weight Proteosomes:weight C.tr./E.coli LPS), which resulted in a ratio of approximately 1:1.8post-dialysis. A mixture of Proteosomes and E. coli LPS prior todialysis was 1:1.35 and was approximately 1:1.4 after dialysis.

In order to determine whether Chlamydia LPS provided in a proteosomeformulation is able to protect mice specifically against a liveChlamydia bacterial challenge, groups of 16 mice (6-8 weeks-old) wereanesthetized and then immunized intranasally on days 0 and 22 withProteosome:C.t./E. coli rLPS. Mice were also treated with Proteosome:E.coli LPS, or Proteosome:Plesiomonas shigelloides LPS. The Proteosome:LPSformulations were given at Proteosome:LPS ratios and doses described inTable 2. Other groups of mice were given either 400 infection units(IFUs) of live CT MoPn (positive control) or HeLa cell extract (negativecontrol; corresponding to the volume of extract required to purify 400IFUs of mouse pneumonitis strain of C. trachomatis (CT MoPn) grown ininfected HeLa cells) as a single dose on day 0. All immunizations weregiven in volumes of 25 μl (12.5 μl/nostril).

Blood was aseptically collected from the retroorbital sinuses of eachmouse on day −1 and day 30. Bronchoalveolar lavages were performed onday 30 on 6 mice per group. On day 34, each of the remaining 10mice/group was given an intranasal challenge with 5,000 IFUs of live CTMoPn. Daily body weights of these mice were measured for 10 consecutivedays. Cardiac exsanguination and lung collection were performed on micesacrificed during this 10-day period. The quantity of Chlamydia IFUs inthe lungs was determined by applying lung and lavage samples to HeLacells and identifying Chlamydia elementary bodies (EB) using fluorescentspecific anti-Chlamydia monoclonal antibodies.

Mice that were immunized nasally with 400 IFUs CT MoPn (group 2 Ct400)were highly protected against body weight loss (BW max loss) (Table 3;P=0.001326). None of the mice in this group had detectable C.trachomatis in their lungs (Table 4; P=4×10⁻⁹) up to ten days afterintranasal challenge with 5,000 IFUs CT MoPn. Control mice that receivedHeLa cell extract (group 1 HELA) were not protected from lethalchallenge as shown by high body weight maximum losses (% reduction) andhigh titers of Chlamydia infection units (IFUs) in the lungs (Tables 3and 4). Mice treated with proteosome preparations containing ChlamydiaLPS, and also either E. coli or P. shigelloides LPS, showed significantprotection against weight loss and bacterial growth (Tables 3 and 4).

To investigate whether protection may be due, at least in part, tospecific anti-Chlamydia LPS antibodies, the presence of Chlamydiaspecific antibodies in sera and lung from immunized mice was determinedby ELISA using inactivated whole cell Chlamydia as the antigen source.Sera were obtained from animals immunized with proteosome-LPS vaccinestwo weeks after the second immunization on Day 22. Sera from animalsimmunized with HeLa cell extract or CT-MoPn were obtained on day 30after the first and only immunization. Results presented in Table 2demonstrated that antibodies present in serum and lung from animalsgiven Proteosomes formulated with Chlamydia LPS bound to whole cellChlamydia.

To determine if the antibodies that bound to Chlamydia cross-reactedwith epitopes present on E. coli LPS and P. shigelloides LPS, binding ofantibodies in sera and lung from treated animals to these LPS types wasalso determined by ELISA. The results presented in Table 2 demonstratedthat antibodies in the sera of mice immunized with each of the specificLPS types in the Proteosome:LPS formulations bound only to thecorresponding LPS. Only antibodies from animals immunized withproteosome-formulated Chlamydia LPS bound to whole cell Chlamydia.Proteosome formulated with LPS from either E. coli alone or P.shigelloides failed to bind to whole cell Chlamydia; however, thesepreparations significantly protected mice against Chlamydia challenge(see Tables 3 and 4).

TABLE 2 Immunogenicity of Protollin Compositions AdministeredIntranasally to BALB/c Mice Serum Anti-LPS Dose Level IgG Ab Titers⁽²⁾Anti-C. tr. MoPn Ratio. C.t./ EB Titers (ng/ml) μg Projuvant:μg E. coliE. coli P. shig. Serum⁽³⁾ Lung⁽⁴⁾ Group Antigens⁽¹⁾ LPS rLPS LPS LPS IgGIgA IgG 1 HeLa cell Not applicable — — — <40 <12 <2 extract 2 Live EBs400 IFUs 1280  — — 10,906 669 173 CT-MoPn 3 E. coli LPS  12:8 160 1280 —<40 378 <2 4 C.t./E. coli 1.5:1 640  40 — 3,158 1532 109 rLPS 5 C.t./E.coli 1.6:8 320 — — 2,187 404 15 rLPS   1:5 6 P. shigella  10:8.6 —— >>2560 <40 <12 <2 LPS   1:1 ⁽¹⁾Intranasal immunizations (25 μl, 12.5μl/nostril) were given once at Day 0 to Group 1 (HELA cells) and group 2(Chlamydia trachomatis)) or given twice, Day 0 (upper ratio) and Day 22(lower ratio) to group 3-6 (proteosome: LPS vaccines) to anesthetizedmice (16 mice/group). ⁽²⁾Anti-LPS antibody titers in sera (pools from 6mice) obtained 5 weeks (day 30) post first immunization (2 weekspost-second immunization with proteosome-LPS vaccines) are expressed asdilution that gave an O.D. at 450 nm approximately twice greater thanthe background. ⁽³⁾No serum anti-Chlamydia IgA was detected independentof the antigen used for immuniziation. ⁽⁴⁾For groups 1 and 2, pools ofhomogenized lungs were analyzed. For groups 3-6, pools of lung lavageswere analyzed. The lungs and lung lavages were obtained on day 30. “—”:No antibody detected.

TABLE 3 Percent Body Weight Reduction (Maximum Loss) Post-SecondTreatment Group Mouse # 1 2 3 4 5 6 in Group HeLa Ct400 Ec12:8 Ct12:8Ct1.6:8 Ps10:9 1 29.3 2 10.6 11.2 11.7 15.5 2 19.4 1.1 32.6 8.7 7.4 1.73 35.5 9.8 15.1 5.8 18 11.4 4 28.6 16.9 16.2 7 20.5 14.2 5 28.3 14.537.7 16.4 7 7 6 30.3 17.6 17.5 11.7 12.3 8.1 7 36.9 6.8 5.9 36.8 4.2 6.28 23.1 3.1 8.4 41.2 7.9 9 25.7 9 8.9 4.5 24.2 9.1 10  26.2 20.9 28.6 2.68.5 13.5 Gmean 27.88 7.89 13.85 8.79 12.56 8.25 t-test log 0.0013260.014 0.000124 0.00224 1.727 × vs HeLa 10⁻⁵

TABLE 4 C. trachomatis IFUs in the Lung Post-Second Treatment Group 1 23 4 5 6 Mouse # in Group HeLa Ct400 Ec12:8 Ct12:8 Ct1.6:8 Ps10:9 1 485009 9 9 9 1100 2 51300 9 91000 9 9 9 3 1100 9 9 9 14900 290 4 6000 9 22509 1500 9 5 200 9 24400 9 9 9 6 35000 9 500 3150 9 9 7 47000 9 9 46500 99 8 1300 9 9 9 29400 320 9 63000 9 9 9 16300 9 10  77000 10 1900 9 95000 Gmean 11304.14 9.10 221.18 38.02 149.81 55.38 t-test log vs HeLa 4× 10⁻⁹ 0.0088 0.00015 0.0050 6.71 × 10⁻⁵ Ct400 - Chlamydia; Ec12:8 - E.coli LPS; Ct12:8 or Ct1.6:8 - Chlamydia/E. coli LPS; and Ps10:9 - P.shigelloides LPS.

Example 15 Duration of Innate Immune Response Protection AgainstChlamydia

The longevity of the nonspecific protection induced by immunostimulatorycompositions, such as Proteosome:LPS (Protollin) or Proteosomes(projuvant), was examined. Groups of 10 mice were treated on day 0 andday 22 with (a) Proteosomes alone (Prot10), (b) Proteosomes formulatedwith LPS from E. coli (Ec10:14); (c) Proteosomes formulated withC.tr./E. coli LPS (Ct10:18); (d) Proteosomes formulated with LPS from P.shigelloides (Ps10:12); (e) live Chlamydia (800 IFUs) (CT800); or (f)HeLa cell mock infection (HeLa). For each treatment, different groups ofmice were challenged with viable Chlamydia bacteria at 2, 5, 8, or 11weeks post-second treatment. Protection was evaluated by determiningmaximum body weight loss (% reduction) and determining Chlamydia IFUs inthe lung for each mouse (designated by Mouse # in Group). The data arepresented in Tables 5-12.

As described in Example 14, treatment with each of the Proteosome:LPSformulations or immunization with live Chlamydia provided significantprotection compared to the HeLa cell control groups as indicated byprevention of weight loss and/or reduction in bacterial titers in thelung. Protection of animals treated with Proteosomes alone lasted forapproximately 5 weeks after the second immunization. An assay todetermine the T cell proliferative response following re-stimulationwith Chlamydia antigen was performed with mouse splenocytes. Spleensfrom each group of mice were pooled and processed into single cellsuspensions according to standard methods. The splenic cell suspensionswere then incubated with different concentrations of Chlamydia antigen.Cytokines (IFN-γ, IL-10, IL-2, and TNF-α) released into culturesupernatants were determined by quantitative ELISA using OptEIA kits (BDBiosciences, San Jose, Calif.). In these experiments Chlamydia-specificsplenic T cell responses induced by the Proteosome:LPS formulations werenot observed in immunized mice. In the absence of Chlamydia-specificantibody or antigen-specific T cell responses in these immunizedanimals, a role for nonspecific, antigen-independent (innate) immunityis suggested as a mechanism for protecting mice from Chlamydia lunginfection. Similarly, in the experiment described in Example 14, anantigen-specific T cell response was observed only in animals thatreceived Chlamydia bacteria and not in animals that received any of theProteosome:LPS formulations.

TABLE 5 Body Weight Maximum Loss Measured after Challenge 2 WeeksPost-Second Immunization Group 1 2 3 4 5 6 Mouse # in Group HeLa CT800Ec10:14 Ct10:18 Ps10:12 Prot10 1 33.6 17 37 23.6 35 30.3 2 34.4 12.717.4 34.1 14.6 15.6 3 41.1 13 23.2 8.3 2.8 31.7 4 36.5 13.9 29.3 8.1 9.438.7 5 29.1 5.8 22 16.7 15.2 25 6 41.1 12.1 30.1 10.8 11.7 26.8 7 38.110.9 12.3 7.6 10.7 15.5 8 34.3 2.9 34.5 8.1 10.2 25.3 9 30.1 21.4 15 4.414.3 19.9 10  30.5 19.2 23.6 39.4 22.6 23.5 G mean 34.64 11.35 23.1112.64 12.38 24.28 t-test log vs. HELA 1.83 × 10⁻⁵ 0.003578 0.0003930.000123 0.00253

TABLE 6 Lung Chlamydia IFUs Quantified after Challenge 2 WeeksPost-Second Immunization Group 1 2 3 4 5 6 Mouse # in Group HeLa CT800Ec10:14 Ct10:18 Ps10:12 Prot10 1 465000 9 368000 41700 5300000 100000 24920000 9 25600 4200000 20000 9 3 1170000 9 725000 9 9 1200000 4 22700009 1510000 9 9 100000 5 497000 9 128000 272000 58100 72400 6 8660000 9124000 69000 9 114000 7 2010000 9 60800 9 9 9 8 273170 9 1180000 9 9437000 9 70100 9 44200 9 27100 33000 10  456000 10 52700 2850000 6950028900 G mean 925084.91 9.10 175269.07 1880.56 962.86 17882.17 t-test logvs. HELA 1.6705 × 10⁻¹⁵ 0.019655 0.00418 0.000736 0.010898 Values of 9and 10 were used for calculations of t-test; however, Chlamydia was notdetected.

TABLE 7 Body Weight Maximum Loss Measured after Challenge 5 WeeksPost-Second Immunization Group 1 2 3 4 5 6 Mouse # in Group HeLa CT800Ec10:14 Ct10:18 Ps10:12 Prot10 1 24.1 12.9 8.9 37.2 20.7 11.6 2 31.720.4 8.2 12.1 28.6 11.3 3 31.5 13.5 32.7 24.9 13.5 19.8 4 39.4 15.5 15.214.5 18.9 26.3 5 40.9 12.1 22.5 26.2 25.8 32.2 6 33.8 4.7 30.2 26.1 24.311.2 7 25.7 28.9 2.5 25.1 15.2 20.9 8 23.3 17.4 38.3 18.5 5.2 15.3 940.1 11.8 24.6 15.9 10.4 37 10  39.4 13.4 13.5 38.9 10.8 8.3 G mean32.31 13.79 15.47 22.41 15.58 17.31 t-test log vs. HELA 5.9122 × 10⁻⁵0.01443 0.018168 0.000717 0.002056

TABLE 8 Lung Chlamydia IFUs Quantified after Challenge 5 WeeksPost-Second Immunization Group 1 2 3 4 5 6 Mouse # in Group HeLa CT800Ec10:14 Ct10:18 Ps10:12 Prot10 1 61200 9 9 573000 29200 9 2 1220000 9 9172000 38900 9 3 4620 9 75600 686000 20000 96900 4 2570000 9 41900 9700026300 217000 5 5910000 9 62300 181000 40000 121000 6 1420000 9 9710000863000 69300 9 7 200000 9 9 157000 82800 52300 8 9 9 7000000 9 9 83300 9538000 9 889000 9 9 339000 10  889000 10 9 409000 9 9 G mean 151583.489.10 6156.61 37381.58 3162.18 2749.09 t-test log vs. HELA 4.179 × 10⁻⁷0.1725 0.4684 0.0456 0.0615 Values of 9 and 10 were used forcalculations of t-test; however, Chlamydia was not detected.

TABLE 9 Body Weight Maximum Loss Measured after Challenge 8 WeeksPost-Second Immunization Group 1 2 3 4 5 6 Mouse # in Group HeLa CT800Ec10:14 Ct10:18 Ps10:12 Prot10 1 35.1 9 23.7 7.5 14 35.6 2 34 10.2 11.610.8 26.9 25.6 3 16.1 7.6 21.2 9.9 12.8 31.7 4 15.2 13.2 8.7 18.1 21.925.2 5 34.7 18.7 15.9 18.1 17.5 9.5 6 36.4 8.2 34.9 6 24.5 20.3 7 32.212.4 10.6 11.7 27.4 33.2 8 33.5 20.8 26.4 26.2 6.7 25.5 9 32.1 10.2 9.75.1 16.7 15.3 10  38.6 13.9 7.7 18.7 14.4 13.6 G mean 29.46 11.78 15.0311.65 16.98 21.84 t-test log vs. HELA 9.657 × 10⁻⁶ 0.00314 0.0002180.00505 0.1013

TABLE 10 Lung Chlamydia IFUs Quantified after Challenge 8 WeeksPost-Second Immunization Group 1 2 3 4 5 6 Mouse # in Group HeLa CT800Ec10:14 Ct10:18 Ps10:12 Prot10 1 6840000 9 11200 9 92400 355000 2 2260009 9 129000 257000 157000 3 9 9 14200 9 9 4260000 4 9 9 3340 137000 577069200 5 2040000 9 57700 352000 15600 6650 6 5.33 × 10⁷ 9 1710000 353018500 198000 7 1210000 10000 9 9 345000 7140000 8 1.44 × 10⁷ 9 2820015300 3040 124500 9 667000 9 9 9 47300 163000 10  2210000 9 9 1880016700 65600 G mean 226134.74 18.15 1255.06 1451.26 14026.81 214441.56t-test log vs. HELA 9.626 × 10⁻⁵ 0.0344 0.0395 0.1809 0.978 Values of 9and 10 were used for calculations of t-test; however, Chlamydia was notdetected.

TABLE 11 Body Weight Maximum Loss Measured after Challenge 11 WeeksPost-Second Immunization Group 1 2 3 4 5 6 Mouse # in Group HeLa CT800Ec10:14 Ct10:18 Ps10:12 Prot10 1 28.3 11.3 11.5 18.2 21.2 16.9 2 19.97.9 21.1 32.7 15.8 22.5 3 29.4 8.6 22.6 25.5 24.8 16.8 4 39.3 13.2 923.3 8.8 33.3 5 30.7 15.7 6.2 23.7 5.3 16.1 6 19 15.1 39.1 38.4 6.8 35.77 38.8 12.4 22.3 25.6 12.5 38 8 34 17.7 15.8 28.6 15.5 27.9 9 19.7 15.524 24.6 18.5 10  27.2 13.3 38.1 26.9 G mean 27.72 12.69 16.70 27.1912.26 24.04 t-test log vs. HELA 3.74 × 10⁻⁶ 0.02195 0.8653 0.0007390.30697

TABLE 12 Lung Chlamydia IFUs Quantified after Challenge 11 WeeksPost-Second Immunization Group 1 2 3 4 5 6 Mouse # in Group HeLa CT800Ec10:14 Ct10:18 Ps10:12 Prot10 1 192000 9 9 2310 29100 9 2 9090 9 153000576000 17500 28300 3 476000 9 338000 20800 17900 5000 4 2225000 9 3800031700 9 2100000 5 2890000 9 8000 5000 9 100000 6 21800 9 2500000 4140009 3270000 7 1.12 × 10⁷ 9 6430 10000 30800 3330000 8 4.17 × 10⁷ 9 1860045000 30000 95500 9 17500 9 37400 11400 10000 10  216000 10 6800001500000 G mean 427705.51 9.10 22671.14 38034.29 1254.65 72783.09 t-testlog vs. HELA 5.44 × 10⁻¹⁰ 0.0597 0.0421 0.00259 0.268 Values of 9 and 10were used for calculations of t-test; however, Chlamydia was notdetected.

Example 16 Protollin Stimulates Protective Innate Immunity AgainstInfluenza Virus Infection Experiment 1

Mice were given a single intranasal dose of Protollin (containingapproximately 5 μg each of Neisseria OMPs and S. flexneri LPS) on day 1,2, or 3 prior to intranasal challenge with 25 LD₅₀ mouse-adapted A/H3influenza virus (Hong Kong). The virus was propagated according tostandard methods (original seed stock was a generous gift from Dr. PhilWyde (Baylor University, Waco, Tex.)). Mice were weighed prior tochallenge and every 2 days after for a total of 14 days. Morbidity wasassessed by weighing individual survivors and expressing weight changeas the percent of weight on the day of challenge (see FIG. 8B). The dayof any death was also recorded (see FIG. 8A). All mice that receivedProtollin 3 days prior to challenge survived and also lacked acutemorbidity (maximum weight loss was 7%). Seventy percent survival wasobserved in the groups of animals that received Protollin 1 or 2 daysprior to challenge; however, animals in both groups suffered 15-18%weight loss. The statistical significance of delay to time of death foreach group as a whole was assessed by the Wilcoxon signed-rank test. Allmice that received Protollin 72 hours prior to challenge survived.Compared to the survival data for the negative control group (noProtollin), survival of mice given Protollin on day 3 prior to challengewas highly significant (P≦0.001; Fisher's Exact Probability test).Seventy percent of mice that received Protollin either 1 or 2 days priorto challenge survived (P≦0.07 compared with the negative control group).While the absolute number of survivors in these two groups was notsignificantly different from the number of survivors in the controlgroup, the time to death was significantly prolonged in both groupscompared to control mice (P≦0.05 or ≦0.01 respectively in the groupsgiven Protollin 1 or 2 days prior to challenge).

Morbidity was monitored in surviving mice in all groups, using loss ofbody weight (relative to the day of challenge) as a surrogate ofmorbidity resulting from infection. All mice lost weight during theperiod of monitoring although the mice given Protollin lost less weightthan control mice given PBS. Weight loss was also dependent on the timebetween Protollin administration and challenge. Mice given Protollin 3days prior to challenge suffered less weight loss than those givenProtollin 2 days before challenge, and the animals given Protollin 2days before challene lost less weight than those given Protollin one daybefore challenge. Until day 8 (after which time the limited numbers ofsurvivors in the control group made statistical comparisons unreliable),control mice lost significantly more weight than mice that receivedProtollin (on days 4 and 6 post challenge, P≦0.001 vs all mice givenProtollin; on day 8 post challenge, P≦0.01 and P≦0.05, respectively, vsmice given Protollin 3 days or 2 days prior to challenge). These resultsshowed that Protollin induced innate responses that protected miceagainst death following lethal, live virus challenge and significantlyreduced morbidity associated with infection.

Experiment 2

In addition, duration of protection within a limiting dose range wasanalyzed. A single dose of 3, 1, or 0.3 μg Protollin was administered togroups of mice (10 animals per group) on day 15, 12, 9, 6, or 3 prior tochallenge with 25 LD₅₀ of a mouse-adapted A/H3 influenza virus. Humaneendpoint indicators for this experiment were based on body weight,appearance, and behavior. Animals were scored from 0-3 in each categoryas follows. For body weight, a score of 0 indicated no loss ofstart-of-study body weight; 1 indicated 10% or less loss ofstart-of-study body weight; 2 indicated 11-19% loss of start-of-studybody weight; and 3 indicated 20% or more loss of start-of-study bodyweight. For appearance, a score of 0 indicated normal appearance; ascore of 1 indicated fur erected; a score of 2 indicated fur erectedoily, nasal and/or ocular discharge; a score of 3 indicated hunchedback, severe dehydration. For behavior, a score of 0 indicated normalbehavior; a score of 1 indicated abnormal gait and weakness; a score of2 indicated activity decreased, severe tremors; and a score of 3indicated inactive. Mice with a score of 3 or more in single or combinedsymptoms were euthanized.

All mice in the control group (no Protollin) and the groups given 0.3 μgProtollin met endpoint criteria six days post-challenge and wereeuthanized. Of the animals receiving 1 μg Protollin, all were euthanized6 days post challenge with the exception of animals in the group dosed 3days prior to challenge, in which 5 mice survived until 8 days postchallenge. Compared to the control group, survival of animals in thisgroup constituted a significant delay in the time to death (P<0.05 forthe group as a whole).

In the groups given 3 μg Protollin, 30% of animals dosed 6 days prior tochallenge survived the study; the remaining 70% met endpoint criteriabetween 6 and 8 days post-challenge. Although these results suggest thatthe number of survivors was not significantly different from the controlgroup (by Fisher's Exact Probability Test), the time to death for thegroup as a whole was significantly different from the control group(P<0.001). Fifty percent of mice receiving 3 μg Protollin, 3 days priorto challenge, survived the study (P<0.05 by Fisher's Exact ProbabilityTest); the other 50% of mice reached endpoint criteria between 6 and 8days post-challenge. Again for the group as a whole, the time to deathwas significantly different from the control group (P<0.001).

In these experiments, the induction of a protective nonspecific,antigen-independent immune response occurred above a threshold range of3-5 μg Protollin, and when Protollin was administered 3-6 days prior tochallenge. Furthermore, co-administration of an influenza antigen(derived from a homotypic variant of the mouse adapted A/H3 influenzastrain used for challenge from) with Protollin did not inhibit theprotective innate immune responses. Induction of a protective innateimmune response was also induced by Protollin comprising another smoothLPS from a Gram-negative bacterium—in this instance a non-pathogenicstrain of E. coli (E. coli 017).

Experiment 3

Groups of mice were given Protollin 8, 6, 4, and 2 days before challengeand on the day of challenge (30 minutes prior to challenge). Othergroups of mice were dosed at the same time with Protollin in combinationwith influenza antigen derived from a homotypic variant virus of themouse adapted A/H3 influenza strain used for challenge. Mice werechallenged with approx 40 LD₅₀ of mouse adapted A/H3 live virus andmonitored for 14 days post challenge as described above.

At the 40 LD₅₀ dose of virus, no animals in the PBS only control groupsurvived. Despite this lethal challenge, 50% of mice that had receivedProtollin 6 days prior to challenge survived (P≦0.05 compared withcontrol mice; Fisher's Exact Probability test). Groups of animals thatreceived Protollin 4-6 days prior to challenge had the greatest percentsurvival. Monitoring changes in body weight (a surrogate of morbidityfollowing infection) in surviving mice indicated that the optimal timefor dosing was approximately 4 days prior to challenge. All mice dosed2, 4, or 6 days prior to challenge lost less body weight than the othermice and began recovering body weight sooner.

Of the mice given Protollin in combination with antigen prior to lethalchallenge, most survivors were in the groups dosed 4 and 6 days prior tochallenge (100% and 60%; P≦0.001 and 0.01, respectively, compared tocontrols). Specific antibody (IgG) responses to the influenza antigenwould be expected to be less than optimal within 4-6 days of receivingantigen. As indicated in the prior experiments, changes in body weightconfirmed that induction of innate immune responses and subsequentprotection against mortality and morbidity occurred when mice were dosedduring a 2-6 day period prior to challenge.

Example 17 Allergen-Induced Mouse Model of Allergic Asthma

This Example describes a mouse allergic asthma animal model. Mice wereexposed to birch pollen extract (BPEx) multiple times to stimulateinflammation and airway hyperresponsiveness (i.e., simulating anallergic reaction). Briefly, six to eight week-old BALB/c mice weresensitized on day 0 by a single intraperitoneal (i.p.) injection with 8μg of BPEx (Greer Laboratories, Inc.) and 1 mg aluminum hydroxide (alum)(Alhydrogel®, Superfos Biosector, Kvistgard, Denmark) in 150 μlphosphate buffered saline (PBS). After sensitization, mice were thenchallenged intranasally (i.n.) under light halothane anesthesia oncedaily on days 15, 16, and 17 with 10 μg BPEx in 36 μl PBS (18 μl pernostril). Controls included (1) sham sensitized mice, who received 150μl PBS i.p. on day 0 and then were challenged i.n. under light halothaneanesthesia once daily on days 15, 16, and 17 with 10 μg BPEx in 36 μlPBS (18 μl per nostril), and (2) sham challenged mice, who weresensitized i.p. on day 0 with 8 μg BPEx and 1 mg alum and then receivedi.n., under light anesthesia, 36 μl PBS (18 μl per nostril) once dailyon days 15, 16, and 17. Eight mice received each type of treatment.After sensitization and challenge, mice were given an intravenous (i.v.)bolus of methacholine (MCh), a bronchoconstrictor, to induce airwayhyperresponsiveness (AHR). Two days after the final challenge (i.e., onday 19), airway responses (respiratory resistance and elastance) to MChtreatment were measured. Additional analyses were performed to assessinflammation.

Example 18 Analysis of Allergen-Induced Mouse Model of Allergic AsthmaAirway Hyperresponsiveness (AHR)

Determination of AHR was performed as follows. BALB/c mice treated asdescribed in Example 17 were sedated by an i.p. injection of xylazinehydrochloride (10 mg/kg) and subsequently anaesthetized with sodiumpentobarbital (30 mg/kg). A small incision was made in the neck toisolate the jugular vein, which was catheterized. A tracheostomy wasperformed, and a tube was inserted into the trachea so that the animalcould be mechanically ventilated. Animals were ventilatedquasi-sinusoidally (inspiratory to expiratory ratio of 1:1) using asmall animal ventilator (FlexiVent; SCIREQ™, Montreal, Canada) with thefollowing settings: a respiratory rate of 150 breaths/min, a tidalvolume of 0.15 ml, and a positive end expiratory pressure (PEEP) levelof 1.5 cm H₂O. Mice received an intravenous injection of pancuroniumbromide (0.5 mg/kg) to induce paralysis so that the animals could bemechanically ventilated. Heart rate was monitored via EKG to ensure thatanimals were deeply anesthetized. Following inflation to airway pressureof 30 cm H₂O to provide a standard volume history, MCh was given via thejugular cannula in doubling doses from 20 to 640 μg/ml. Respiratorysystem resistance and elastance were measured during the oscillationequal to those used during mechanical ventilation before administrationof MCh and repeated every 15 seconds after delivery of MCh, with peakvalues reported. The airway resistance (R_(L)) measurement provides aquantitative assessment of the level of constriction in the lungs—thatis, an increase in airway resistance represents an increase of airwayobstruction, which may be caused by an inflammatory response. The airwayelastance (E_(L)) is a measure of the elastic rigidity of the lung;therefore, increased elastance values indicate an increased stiffness ofthe lungs. R_(L) and E_(L) were calculated with software provided by theFlexiVent manufacturer using multiple linear regression to obtain thebest fit for the following equation:

P=P _(res) +P _(el) +P _(in) =FR _(L) +VE _(L) +K

in which P is gas pressure applied by the mechanical respirator; P_(res)is resistive pressure; P_(el) is elastic pressure; P_(in) is an inertivepressure; F is flow of gas; V is lung volume relative to functionalresidual capacity; and K is a constant (Irvin et al., Respir. Res. 4:4(2003)). Airway resistance and airway elastance are presented as meanvalues±SEM. Student's t-test was used to determine the level ofdifference between animal groups.

Serum

Immediately following measurements of airway responsiveness, mice weresacrificed by exsanguination via cardiac puncture, the collected bloodwas centrifuged, and the resulting serum was transferred to a clean tubeand frozen. The sera were analyzed using ELISAs to determine whetherBPEx-specific antibodies were present.

Bronchoalveolar Lavages (BALs) and Eosinophilia

Following exsanguination, the descending aorta was cut and the heart wasperfused with 5 ml saline buffer to remove blood from the lungs prior toperforming BALs. A total of 4.6 ml saline buffer was instilled through atracheostomy canula in an initial 0.6 ml volume followed by 4 successive1 ml volumes. The return from the first 0.6 ml of lavage fluid wascentrifuged, and the supernatant was analyzed by ELISA to detectantibodies and cytokines.

The cells harvested from the initial lavage fluid were resuspended insaline buffer and then pooled with cells recovered by centrifugationfrom the subsequent four aliquots of lavage fluid. Total cell numberswere counted by using trypan blue stain and a hemocytometer. Thecytospin slides of BAL cells were prepared using a cytocentrifuge(Cytospin model II; Shandon, Pittsburgh, Pa.).

Eosinophilia was evaluated in BALs by measuring the percent ofdifferentially stained macrophages, eosinophils, neutrophils,lymphocytes, and epithelial cells (Diff-Quick, International MedicalEquipment) in the lavage samples. Differential cell counts weredetermined by light microscopy from a count of at least 200 cells.

Lung Tissue

Following BALs, the lungs of each mouse are exposed and the left lobe isclamped. The largest lobe of the right lung is put directly into 10%paraffin. The second largest lobe is put in an RNA extraction solution(Rneasy® RLT buffer; Qiagen Inc, Mississauga, Ontario), which is kept at4° C. overnight and then stored frozen at −70° C. for subsequent use forreal-time quantitative polymerase chain reaction (QPCR). The two otherlobes of the right lung are transferred to an Eppendorf tube, immersedin liquid nitrogen, and then stored at −70° C. Lung homogenates areprepared and supernatants are analyzed by ELISA for BPEx-specific andtotal antibodies and for cytokines levels. The left lung is inflatedwith 5% optimal cutting temperature (OCT) embedding compound (MilesLabs, Elkhart, Ind.) (approximately 25 cm pressure), put in 100% OCTwith immersion in isopentanol (beaker in liquid nitrogen; i.e., snapfrozen), and stored at −70° C.

Lung tissues in paraffin are sliced, and sections are stained withperiodic acid-Schiff (PAS staining) for evaluation of mucus production.Paraffin sections are also analyzed for the presence of collagen (VanGieson staining) and eosinophils (Giemsia staining). In addition, airwaydamage is assessed in the stained sections. Frozen lung tissues in OCTembedding compound are sliced and analyzed by in situ immunostaining.Eosinophils are quantified by immunostaining with an anti-mouse majorbasic protein (MBP) antibody. Lung sections are used for theidentification of cytokines (anti-IL-4, anti-IL-5, or anti-IFN-γ),T-cells (anti-CD3), and macrophages (anti-CD68).

ELISAs of Antibody and Cytokine Levels

ELISAs are used to identify specific antibodies (IgA, IgE, IgGI, andIgG2a) and specific cytokines (IL-4, IL-5, IL-10, IL-13, TNF-α, andIFN-γ). Sera, BALs, and lung homogenates are analyzed for BPEx-specificand total IgE using the OptEIA™ mouse IgE set (BD Pharmingen,Mississauga, Ontario). Sera, BALs, and lung homogenates are analyzed forBPEx-specific and total IgG1 and IgG2a using reagents from SouthernBiotech Associates, Inc. (Birmingham, Ala.). Sera, BALs, and lunghomogenates are analyzed for BPEx-specific and total IgA using reagentsfrom Bethyl Laboratories, Inc. (Montgomery, Tex.). BALs and lunghomogenates are analyzed for the level of IL-4, IL-5, IL-10, TNF-α, andIFN-γ using reagents of BD Pharmingen (Mississauga, Ontario). BALs andlung homogenates are analyzed for the level of IL-13 using reagents ofR&D Systems (Minneapolis, Minn.). Antibody and cytokine titers areexpressed as ng/ml and pg/ml, respectively, deduced from standards runin parallel with corresponding recombinant antibodies or cytokines.

Quantification of IL-4, IL-5, IL-10, IL-13, TNF-α, and IFN-γ byreal-time, QPCR in lung samples kept frozen in RNA extraction solution(as described herein) are initiated by isolation of total cellular RNAusing the Qiagen RNeasy® Mini Kit (QIAgen® Inc.). The concentration ofthe RNA extracted is determined by measuring optical densities at 260 nm(OD₂₆₀), and purity is evaluated based on OD₂₆₀/OD₂₈₀ ratios equal to orgreater than 1.8. Reverse transcription is performed on 1 μg RNA samplesusing Omniscript™ reverse transcriptase kits (QIAgen® Inc.) in aconstant volume of 20 μl. A 1 μl volume from the resulting complementaryDNA (cDNA) solutions is used for real-time QPCR reactions, which areperformed on a LightCycler™ (Roche Diagnostics, Mannheim, Germany). Thereactions include Sybr®Green I as a double-strand DNA-specific bindingdye in the LightCycler™—primer set (Search Lc, Heidelberg, Germany) fora specific cytokine, or for the S9 ribosomal protein (control,house-keeping gene).

Airway hyperresponsiveness, as measured by both airway resistance (Table13) and airway elastance (Table 14), increased in animals that receivedincreasing quantities of MCh and that were sensitized and challengedwith BPEx compared with mice that were either only sensitized or onlychallenged with BPEx. Following an intravenous injection of MCh at 320μg/ml, sensitized/challenged mice had airway resistance (12.88 cmH₂O.sec/ml) and elastance (103.08 cm H₂O/ml) that were 2-fold higherthan in sensitized/sham mice (p=0.014 and p=0.038, respectively) andsham/challenged mice (p=0.042 and p=0.084, respectively) (see Tables 13and 14). Sensitized/challenged mice, following i.v. injection of MCh at640 μg/ml, showed airway resistance of 36.63 cm H₂O.sec/ml and elastanceof 359.88 cm H₂O/ml, which was 3- and 5-fold higher than in control micethat were only sensitized with BPEx (p=0.014 and p=0.028, respectively)and mice that were only challenged with BPEx (p=0.031 and p=0.062,respectively), respectively (see Tables 13 and 14).

TABLE 13 Mean Respiratory System Resistance (cm H₂O · s/ml) in MouseAllergic Asthma Model Mch (μg/ml) baseline 10 20 40 80 160 320 640 Group1: 0.66 0.68 0.73 0.90 1.59 4.24 12.88 36.63 sens/chal Group 2: 0.690.70 0.75 0.93 1.40 2.58 5.67 10.03 sens/sham Group 3: 0.69 0.71 0.780.97 1.63 3.01 6.11 8.19 sham/chal Data were analyzed by t-test: Group 1vs Group 2 at 320 μg/ml MCh: p = 0.014 Group 1 vs Group 3 at 320 μg/mlMCh: p = 0.042 Group 1 vs Group 2 at 640 μg/ml MCh: p = 0.014 Group 1 vsGroup 3 at 640 μg/ml MCh: p = 0.031

TABLE 14 Mean Respiratory System Elastance (cm H₂O)/ml) in MouseAllergic Asthma Model Mch (μg/ml) base- line 10 20 40 80 160 320 640Group 1: 28.89 30.25 31.89 33.99 37.42 47.52 103.08 359.88 sens/chalGroup 2: 27.63 28.74 30.14 31.70 34.29 40.54 53.08 71.31 sens/sham Group3: 28.48 29.84 31.43 33.58 37.34 44.38 57.82 64.21 sham/chal Data wereanalyzed by t-test: Group 1 vs Group 2 at 320 μg/ml MCh: p = 0.038 Group1 vs Group 3 at 320 μg/ml MCh: p = 0.084 Group 1 vs Group 2 at 640 μg/mlMCh: p = 0.028 Group 1 vs Group 3 at 640 μg/ml MCh: p = 0.062

In addition, the combination of sensitization and challenge with BPEx inBALB/c mice resulted in eosinophilia. Macrophages, neutrophils,eosinophils, lymphocytes, and epithelial cells were enumerated inbronchoalveolar lavage samples (BALs). The percent of each cell type pertotal number of cells is presented in Table 15. In these mice, thepercentage of eosinophils (8.26%) and lymphocytes (10.16%) was 12- and4-fold higher, respectively, than in control mice that were onlysensitized with BPEx (p=0.036 and p=0.024, respectively); these valueswere 3- and 2-fold higher, respectively, than in mice that werechallenged only with BPEx (p=0.204 and p=0.320, respectively)

TABLE 15 Differential Cell Counts (%) in BALs in Mouse Allergic AsthmaModel Macro- Neutro- Epithelial Cell Type phages phils EosinophilsLymphocytes Cells Group 1: 74.13 2.90 8.26 10.16 4.64 sens/chal Group 2:86.42 3.12 0.66 2.34 6.36 sens/sham Group 3: 73.08 7.01 2.56 5.91 6.61sham/chal Data were analyzed by t-test.

Example 19 Protollin-Induced Suppression of Airway Hyperresponsivenessand Airway Inflammation

The allergic asthma mouse model (as described in Example 18) was used toanalyze compositions for suppressing an inflammatory immune response andairway hyperresponsiveness (i.e., suppressing an allergic reaction).Briefly, six to eight-week old BALB/c mice were sensitized i.p. on day 0with 8 μg of BPEx and 1 mg alum in 150 μl PBS. On days 7, 10, and 13after sensitization, groups of eight mice were each immunized i.n. with10 μl (5 μl per nostril) solutions of (1) PBS; (2) 10 μg BPEx; (3) 10 μgBPEx mixed with 10 μg Protollin; or (4) 10 μg Protollin alone. Afterimmunization, the mice were then challenged i.n. under light halothaneanesthesia once daily on days 15, 16, and 17 with 10 μg BPEx in 36 μlPBS (18 μl per nostril). Two days after the final challenge (i.e., onday 19), mice were given an i.v. bolus of MCh (20-640 μg/ml). Airwayresponses (respiratory resistance and elastance) to MCh treatment,inflammation, and eosinophilia were determined as described in Example18.

Sensitized mice that were immunized with a composition comprising BPExwith Protollin or Protollin alone and subsequently challenged i.n. withBPEx, showed reduced airway resistance and elastance in AHR measurementsas intravenous quantities of MCh were increased (see Tables 16 and 17).Following an intravenous injection of MCh at 640 μg/ml, mice treatedwith BPEx mixed with Protollin had reduced airway resistance (12.54 cmH₂O.sec/ml) and elastance (99.73 cm H₂O/ml) by approximately 43% and48%, respectively, compared with mice treated with only PBS (p=0.028 andp=0.050, respectively) or only BPEx (p=0.132 and p=0.220, respectively).Similarly, Protollin adjuvant alone also lowered airway resistance (9.71cm H₂O.sec/ml) and elastance (69.24 cm H₂O/ml) by approximately 56% and64%, respectively, compared to mice treated with only PBS (p=0.005 andp=0.009, respectively) or only BPEx (p=0.029 and p=0.074, respectively).

TABLE 16 Mean Respiratory System Resistance (cm H₂O · s/ml)Post-Administration of Protollin: Birch Pollen Extract (BPEx) in MouseAllergic Asthma Model Mch (μg/ml) baseline 20 40 80 160 320 640 Group 1:PBS 0.61 0.68 0.87 1.48 3.27 9.72 24.13 Group 2: BPEx 0.67 0.71 0.901.47 3.41 8.81 19.87 Group 3: 0.63 0.68 0.79 1.17 2.39 6.03 12.54 BPEx:Protollin Group 4: 0.60 0.64 0.75 1.06 2.00 4.59 9.71 Protollin Datawere analyzed by t-test: Group 3 vs Group 1 at 640 μg/ml MCh: p = 0.028Group 3 vs Group 2 at 640 μg/ml MCh: p = 0.132 Group 4 vs Group 1 at 640μg/ml MCh: p = 0.005 Group 4 vs Group 2 at 640 μg/ml MCh: p = 0.029

TABLE 17 Mean Respiratory System Elastance (cm H₂O)/ml)Post-Administration of Protollin: Birch Pollen Extract (BPEx) in MouseAllergic Asthma Model Mch (μg/ml) baseline 20 40 80 160 320 640 Group 1:PBS 26.33 28.07 30.29 35.05 43.20 75.16 205.50 Group 2: BPEx 25.61 27.2329.27 32.33 40.91 75.15 180.82 Group 3: 24.91 26.56 28.21 30.81 35.5048.69 99.73 BPEx: Protollin Group 4: 24.77 26.15 27.98 30.11 34.83 45.5069.24 Protollin Data were analyzed by t-test: Group 3 vs Group 1 at 640μg/ml MCh: p = 0.050 Group 3 vs Group 2 at 640 μg/ml MCh: p = 0.220Group 4 vs Group 1 at 640 μg/ml MCh: p = 0.009 Group 4 vs Group 2 at 640μg/ml MCh: p = 0.074

The extent of airway inflammation in animals was determined byenumerating immune cells present in bronchoalveolar lavage (BAL) samplesfrom mice treated with PBS, BPEX, BPEX+Protollin, and Protollin alone.The data are presented in Table 18. Animals that were treated with theallergen BPEx mixed with Protollin had reduced levels of BALeosinophils, approximately 56% and 43%, compared to mice that weretreated with only PBS (p=0.36) or with only BPEx (p=0.29), respectively.Treatment with BPEx plus Protollin also reduced the number oflymphocytes in BAL by approximately 51% and 40% compared to animalstreated with PBS only (p=0.04) or BPEx alone (p=0.26), respectively.Animals treated with Protollin alone had reduced levels of BALeosinophils, approximately 71% and 63%, compared with the number ofeosinophils from mice treated with only PBS (p=0.28) or BPEx alone(p=0.14), respectively. Treating mice with Protollin alone also reducedthe number of lymphocytes, approximately 45% and 32% compared with micetreated with PBS only (p=0.10) or BPEx (p=0.40), respectively. Lungsamples were analyzed for the presence of Goblet cells. Reduction inairway inflammation was also indicated by the lower percent of mice thathad mucous-producing Goblet cells in the bronchioles in the grouptreated with BPEx plus Protollin or Protollin alone (29%; that is, 2mice out of 7 mice in each group had at least 1% Goblet cells of thetotal number of bronchial epithelial cells) compared with mice treatedwith PBS only (75%; 5 of 7 mice) or PBEx only (50%; 3 of 6 mice).

Antibodies present in sera that specifically bound to BPEx were detectedby ELISA using the method described in Example 18. BPEx-specific IgE andIgGI were measured at low levels in mouse sera while BPEx-specific serumIgG2a was poorly detectable (see Table 19). Intranasal treatment ofanimals with Protollin alone reduced the levels of BPEx-specific IgE andIgGI by at least 50% compared with mice treated with only PBS or onlyBPEx.

TABLE 18 Airway Inflammation: Immune Cells in Bronchoalveolar Lavage andPercent Mice with Mucus Producing Goblet Cells Macrophages/ EpithelialGoblet Monocytes Neutrophils Eosinophils Lymphocytes cells cellsTreatment (×10⁴/ml) (×10⁴/ml) (×10⁴/ml) (×10⁴/ml) (×10⁴/ml) (% mice) PBS83.0 9.1 14.7 9.4 18.5 71% BPEx 93.6 7.0 11.4 7.7 15.0 50%BPEx/Protollin 66.0 5.4 6.5 4.6 10.4 29% Protollin 92.7 4.9 4.2 5.2 14.429%

TABLE 19 Allergen Specific Antibody Levels in Sera Treatment IgE (×100ng/ml) IgG1 (×1000 ng/ml) PBS 80.77 28.800 BPEx 27.28 15.945BPEx/Protollin 31.53 35.839 Protollin 9.18 7.931

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for eliciting a nonspecific immune response, comprisingadministering to a subject an immunostimulatory composition in an amountsufficient to elicit a nonspecific immune response, wherein theimmunostimulatory composition comprises Proteosomes and liposaccharide.2. The method according to claim 1 wherein the immunostimulatorycomposition is administered by a route selected from at least one ofmucosal, enteral, parenteral, transdermal, transmucosal, nasal, andinhalation.
 3. The method according to claim 2 wherein theimmunostimulatory composition is administered nasally.
 4. The methodaccording to claim 1 wherein the liposaccharide final content by weightas a percentage of Proteosome protein ranges from about 1% to 500%. 5.The method according to claim 1 wherein the Proteosomes andliposaccharide are obtained from the same Gram-negative bacterialspecies.
 6. The method according to claim 1 wherein the Proteosomes areobtained from a first Gram-negative bacterial species and theliposaccharide is obtained from a second Gram-negative bacterialspecies.
 7. The method according to claim 1 wherein the liposaccharideis obtained from a Gram-negative bacterium selected from at least one ofShigella species, Chlamydia species, Yersinia species, Pseudomonasspecies, Plesiomonas species, Escherichia species, Porphyromonasspecies, and Salmonella species.
 8. The method according to claim 1wherein the Proteosomes are obtained from Neisseria species.
 9. Themethod according to claim 1 wherein the Proteosomes are obtained fromNeisseria meningitidis, and the liposaccharide is obtained from Shigellaflexneri.
 10. The method of claim 1 wherein the method further comprisesadministering to the subject an immunogenic composition afteradministering the immunostimulatory composition, wherein the immunogeniccomposition comprises Proteosomes, liposaccharide, and a microbialantigen.
 11. The method according to claim 10 wherein the immunogeniccomposition comprises at least two microbial antigens.
 12. The methodaccording to claim 10 wherein the microbial antigen is a viral antigen,a bacterial antigen, a fungal antigen, or a parasitic antigen.
 13. Themethod according to claim 11 wherein the at least two microbial antigensare obtained from the same microorganism, wherein the microorganism is abacterium, a virus, a fungus, or a parasite.
 14. The method according toclaim 11 wherein the at least two microbial antigens are obtained fromdifferent microorganisms.
 15. The method according to claim 10 whereinthe ratio of the weight of Proteosomes and liposaccharide of theimmunogenic composition to the weight of the microbial antigen of theimmunogenic composition is within a range from 4:1 to 1:4.
 16. Themethod according to claim 10 wherein the ratio of the weight ofProteosomes and liposaccharide of the immunogenic composition to theweight of the microbial antigen of the immunogenic composition is withina range from 1:1 to 1:500.
 17. The method according to claim 10 whereinthe ratio of the weight of Proteosomes and liposaccharide of theimmunogenic composition to the weight of the microbial antigen of theimmunogenic composition is within a range from 1:1 to 1:200.
 18. Themethod according to claim 10 wherein the microbial antigen isrecombinant.
 19. The method according to claim 10 wherein the microbialantigen is a bacterial antigen.
 20. The method according to claim 19wherein the bacterial antigen is obtained from Bacillus anthracis,Chlamydia trachomatis, Yersinia pestis, or Enteropathogenic Escherichiacoli.
 21. The method according to claim 20 wherein the bacterial antigenis Protective Antigen from Bacillus anthracis.
 22. The method accordingto claim 10 wherein the microbial antigen of the immunogenic compositionis a viral split antigen.
 23. The method according to claim 22 whereinthe viral split antigen is an influenza split antigen.
 24. The methodaccording to claim 10 wherein the immunogenic composition isadministered about one to about ten days after the immunostimulatorycomposition.
 25. The method according to claim 10 wherein theimmunogenic composition elicits an adaptive immune response.
 26. Themethod according to claim 1 wherein the nonspecific immune response isan innate immune response that prevents or treats a microbial infection.27. The method according to claim 26 wherein the microbial infection isa viral, parasitic, fungal, or bacterial infection.
 28. The methodaccording to either claim 1 or claim 10 wherein at least one of theimmunostimulatory composition and the immunogenic composition furthercomprises a pharmaceutically acceptable carrier.
 29. The methodaccording to claim 26 wherein the microbial infection is a viralinfection.
 30. The method according to claim 29 wherein the viralinfection is an influenza viral infection.